Compositions and methods for cellular processing

ABSTRACT

Provided herein are compositions and methods for cellular analysis. Nucleic acid from single cells may be processed in one or more partitions. A partition may comprise a cell bead and one or more enzymes for nucleic acid processing. A partition may comprise a functionalized polymer. In some cases, single cells may be subjected to epigenetic analysis, thereby generating an epigenetic profile for each cell from a plurality of cells.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/845,164, filed May 8, 2019, and U.S. ProvisionalPatent Application No. 62/720,065, filed Aug. 20, 2018, each of whichapplications is entirely incorporated herein by reference.

BACKGROUND

A sample may be processed for various purposes, such as identificationof a type of moiety within the sample. The sample may be a biologicalsample. Biological samples may be processed, such as for detection of adisease (e.g., cancer) or identification of a particular species. Thereare various approaches for processing samples, such as polymerase chainreaction (PCR) and sequencing.

Biological samples may be processed within various reactionenvironments, such as partitions. Partitions may be wells or droplets.Droplets or wells may be employed to process biological samples in amanner that enables the biological samples to be partitioned andprocessed separately. For example, such droplets may be fluidicallyisolated from other droplets, enabling accurate control of respectiveenvironments in the droplets.

Biological samples in partitions may be subjected to various processes,such as chemical processes or physical processes. Samples in partitionsmay be subjected to heating or cooling, or chemical reactions, such asto yield species that may be qualitatively or quantitatively processed.

SUMMARY

In some aspects, the present disclosure provides a method for nucleicacid analysis, comprising: generating a partition comprising (i) a cellbead comprising a nucleic acid, (ii) a nucleic acid barcode molecule,and (iii) a polymer; and (b) using said nucleic acid or a fragmentthereof and said nucleic acid barcode molecule to perform one or morereactions in said partition. In some embodiments, the partition is adroplet. In some embodiments, the partition is a well. In someembodiments, the one or more reactions are performed outside the cellbead. In some embodiments, the one or more reactions comprise nucleicacid extension. In some embodiments, the one or more reactions comprisenucleic acid amplification. In some embodiments, the method furthercomprises releasing the barcoded nucleic acid from the partition. Insome embodiments, the cell bead or the polymer is functionalized. Insome embodiments, the polymer is a charged polymer. In some embodiments,the polymer is positively charged. In some embodiments, the polymer isnegatively charged. In some embodiments, the cell bead or the polymer isattached to a reagent. In some embodiments, the reagent is an enzyme, anucleic acid molecule comprising a capture sequence, an aptamer, achemical compound, or a polypeptide. In some embodiments, the reagentcomprises biotin. In some embodiments, the reagent is a small moleculeinhibitor. In some embodiments, the small molecule inhibitor isdeoxythymidine 3′,5′-bisphosphate. In some embodiments, the chemicalcompound is a chelating agent. In some embodiments, the chelating agentis ethylenediaminetetraacetic acid (EDTA) or ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). Insome embodiments, the capture sequence is a poly-thymine (poly-T)sequence, a random N-mer sequence, or a targeted capture sequence. Insome embodiments, the enzyme is a nuclease, transposase, polymerase,helicase, reverse transcriptase, ligase, or phosphatase. In someembodiments, the nucleic acid molecule comprising the capture sequencefurther comprises a functional sequence selected from the groupconsisting of a barcode sequence, a unique molecular index (UMI)sequence, a sequencing primer sequence, a partial sequencing primersequence, and a sequence configured to attach to the flow cell of asequencer. In some embodiments, the partition further comprises anenzyme configured to perform the one or more reactions on the nucleicacid molecule or a derivative thereof. In some embodiments, thepartition comprises a cation for activating the enzyme. In someembodiments, the cation is magnesium or calcium. In some embodiments,the method further comprises, prior to (a), contacting the cell beadwith the enzyme and performing one or more reactions on the nucleic acidmolecule. In some embodiments, the enzyme is a nuclease, transposase,polymerase, helicase, reverse transcriptase, ligase, or phosphatase. Insome embodiments, the nuclease is micrococcal nuclease (MNase). In someembodiments, the nuclease is DNase.

In some embodiments, the method further comprises, prior to (b), usingthe nuclease to cleave the nucleic acid molecule to generate a nucleicacid fragment. In some embodiments, the enzyme is an engineered enzyme.In some embodiments, the engineered enzyme is an engineered nuclease. Insome embodiments, the engineered nuclease comprises a binding moietyconfigured to bind to a binding partner. In some embodiments, binding ofthe binding partner to the binding moiety inhibits the engineerednuclease. In some embodiments, the binding partner is a protein. In someembodiments, the binding moiety is an aptamer. In some embodiments, thebinding moiety comprises biotin. In some embodiments, the bindingpartner is streptavidin or avidin. In some embodiments, the one or morereactions comprises using a ligase to ligate the nucleic acid barcodemolecule to the nucleic acid molecule or derivative thereof. In someembodiments, the nucleic acid barcode molecule is attached to a bead. Insome embodiments, the bead comprises chemical cross-linkers. In someembodiments, the chemical cross-linkers are disulfide bonds. In someembodiments, the bead is a gel bead. In some embodiments, the nucleicacid barcode molecule is attached to the bead via a releasable linker.In some embodiments, the releasable linker is a disulfide bond. In someembodiments, the nucleic acid barcode molecule is released from the beadprior to performing the one or more reactions. In some embodiments, thepolymer is a linear polymer. In some embodiments, the polymer is abranched polymer. In some embodiments, the polymer is not cross-linked.In some embodiments, the polymer is dextran, polyethylene glycol (PEG),or polyacrylamide.

In some embodiments, the partition further comprises a particle. In someembodiments, the particle is a magnetic particle. In some embodiments,the cell bead comprises the magnetic particle. In some embodiments, theparticle comprises, attached thereto, a nucleic acid molecule comprisinga capture sequence. In some embodiments, the capture sequence is apoly-T sequence, a random N-mer sequence, or a targeted capturesequence. In some embodiments, the polymer is incapable of diffusinginto the cell bead. In some embodiments, the method further comprises,prior to (a), generating a cell bead comprising an analyte carriercomprising the nucleic acid molecule. In some embodiments, the methodfurther comprises lysing the analyte carrier. In some embodiments, theanalyte carrier is lysed prior to (a). In some embodiments, the analytecarrier is lysed subsequent to (a). In some embodiments, the analytecarrier is permeabilized. In some embodiments, the analyte carrier ispermeabilized prior to (a). In some embodiments, the analyte carrier ispermeabilized subsequent to (a). In some embodiments, the analytecarrier is a cell or cell nucleus.

In some aspects, described herein is a composition for nucleic acidanalysis comprising a partition comprising (i) a cell bead comprising anucleic acid molecule, (ii) a barcoded bead comprising a nucleic acidbarcode molecule, and (iii) a polymer. In some embodiments, the cellbead or the polymer is attached to a reagent. In some embodiments, thereagent is an enzyme, a nucleic acid molecule comprising a capturesequence, a chemical compound, a chelating agent, or a polypeptide. Insome embodiments, the reagent comprises biotin. In some embodiments, thereagent is a small molecule inhibitor. In some embodiments, the smallmolecule inhibitor is deoxythymidine 3′,5′-bisphosphate. In someembodiments, the chelating agent is EDTA or EGTA. In some embodiments,the nucleic acid molecule comprising the capture sequence furthercomprises a functional sequence selected from the group consisting of abarcode sequence, a unique molecular index (UMI) sequence, a sequencingprimer sequence, a partial sequencing primer sequence, and a sequenceconfigured to attach to the flow cell of a sequencer. In someembodiments, the capture sequence is a poly-T sequence, a random N-mersequence, or a targeted capture sequence. In some embodiments, thepartition further comprises an enzyme configured to perform the one ormore reactions on the nucleic acid molecule or a derivative thereof. Insome embodiments, the partition further comprises a cation foractivating the enzyme. In some embodiments, the cation is magnesium orcalcium. In some embodiments, the enzyme is a nuclease, a transposase, apolymerase, a helicase, a reverse transcriptase, a ligase, or aphosphatase. In some embodiments, the enzyme is a nuclease. In someembodiments, the nuclease is MNase. In some embodiments, the nuclease isDNase. In some embodiments, the enzyme is an engineered enzyme. In someembodiments, the engineered enzyme is an engineered nuclease. In someembodiments, the engineered nuclease is an engineered MNase. In someembodiments, the engineered nuclease comprises a binding moietyconfigured to bind to a binding partner. In some embodiments, thebinding partner is a protein. In some embodiments, the binding proteinis an aptamer. In some embodiments, the binding moiety comprises biotin.In some embodiments, the binding partner is streptavidin or avidin. Insome embodiments, the polymer is incapable of diffusing into the cellbead. In some embodiments, the polymer is a linear polymer. In someembodiments, the polymer is a branched polymer. In some embodiments, thepolymer is not cross-linked. In some embodiments, the polymer isdextran, polyethylene glycol (PEG), or polyacrylamide. In someembodiments, the partition further comprises a particle. In someembodiments, the particle is a magnetic particle. In some embodiments,the cell bead comprises the magnetic particle. In some embodiments, theparticle comprises, attached thereto, a nucleic acid molecule comprisinga capture sequence. In some embodiments, the capture sequence is apoly-T sequence, a random N-mer sequence, or a targeted capturesequence. In some embodiments, the cell bead comprises a lysed analytecarrier. In some embodiments, the cell bead comprises a permeabilizedanalyte carrier. In some embodiments, the analyte carrier is a cell orcell nucleus.

Disclosed herein, in some aspects, is a method for generating a barcodednucleic acid, comprising: (a) generating a cell bead comprising apermeabilized nucleus and a nuclease; (b) co-partitioning (i) the cellbead, (ii) a nucleic acid barcode molecule, and (iii) a polymercomprising an inhibitor of the nuclease into a partition; (c) using thenuclease to fragment a nucleic acid from the nucleus, thereby generatinga nucleic acid fragment; (d) releasing (i) the nucleic acid fragment and(ii) the nuclease from the cell bead, thereby inhibiting the nucleasewith the inhibitor; and (e) using the nucleic acid fragment and thenucleic acid barcode molecule to generate the barcoded nucleic acid. Insome embodiments, the nuclease is attached to a binding moiety. In someembodiments, the nuclease is attached to the binding moiety via abinding protein. In some embodiments, the nuclease is attached to thebinding protein via a linker. In some embodiments, the binding proteinis Protein A or Protein G. In some embodiments, the nuclease is attachedto the binding moiety via a linker. In some embodiments, the bindingmoiety is an antibody or antibody fragment. In some embodiments, themethod further comprises releasing the barcoded nucleic acid moleculefrom the partition. In some embodiments, the method further comprisessequencing the barcoded nucleic acid molecule. In some embodiments, thepartition further comprises a particle. In some embodiments, theparticle is a magnetic particle. In some embodiments, the particlecomprises, attached thereto, a nucleic acid molecule comprising acapture sequence. In some embodiments, the capture sequence is a poly-Tsequence, a random N-mer sequence, or a targeted capture sequence. Insome embodiments, the partition further comprises a bead. In someembodiments, the bead comprises chemical cross-linkers. In someembodiments, the chemical cross-linkers are disulfide bonds. In someembodiments, the bead is a gel bead. In some embodiments, the nucleicacid barcode molecule is attached to the bead. In some embodiments, thenucleic acid barcode molecule is attached to the bead via an acryditemoiety. In some embodiments, the nucleic acid barcode molecule isattached to the bead via a releasable linker. In some embodiments, thereleasable linker is a disulfide bond. In some embodiments, the nucleicacid barcode molecule is released from the bead prior to (e).

In some embodiments, the polymer is a linear polymer. In someembodiments, the polymer is a branched polymer. In some embodiments, thepolymer is not cross-linked. In some embodiments, the polymer isdextran, polyethylene glycol (PEG), or polyacrylamide. In someembodiments, the polymer is incapable of diffusing into the cell bead.In some embodiments, the inhibitor is an enzyme, a nucleic acid moleculecomprising a capture sequence, a chemical compound, a chelating agent,or a polypeptide. In some embodiments, the inhibitor comprises biotin.In some embodiments, the inhibitor is a small molecule inhibitor. Insome embodiments, the small molecule inhibitor is deoxythymidine3′,5′-bisphosphate. In some embodiments, the chelating agent is EDTA orEGTA. In some embodiments, the nuclease is an engineered nuclease. Insome embodiments, the engineered nuclease is an engineered MNase. Insome embodiments, the engineered nuclease is an engineered DNase. Insome embodiments, the engineered nuclease comprises a binding moietyconfigured to bind to a binding partner. In some embodiments, thebinding partner is a protein. In some embodiments, the binding partneris an aptamer. In some embodiments, the binding moiety comprises biotin.In some embodiments, the binding partner is streptavidin or avidin. Insome embodiments, the polymer is incapable of diffusing into the cellbead. In some embodiments, the partition comprises a cation foractivating the nuclease. In some embodiments, the cation is magnesium orcalcium.

In some aspects, present disclosure provides a composition for nucleicacid analysis comprising a partition comprising (i) a cell beadcomprising a nucleic acid, (ii) a barcoded bead comprising a nucleicacid barcode molecule, and (iii) a polymer.

In some aspects, present disclosure provides a method for generating abarcoded nucleic acid, comprising: (a) generating a cell bead comprisinga permeabilized nucleus and a nuclease; (b) co-partitioning (i) saidcell bead, (ii) a nucleic acid barcode molecule, and (iii) a polymercomprising an inhibitor of said nuclease into a partition; (c) usingsaid nuclease to fragment a nucleic acid from said nucleus, therebygenerating a nucleic acid fragment; (d) releasing (i) said nucleic acidfragment and (ii) said nuclease from said cell bead into said partition,thereby inhibiting said nuclease; and (e) releasing (i) said nucleicacid fragment and (ii) said nuclease from said cell bead into saidpartition, thereby inhibiting said nuclease.

Another aspect of the present disclosure provides a non-transitorycomputer readable medium comprising machine executable code that, uponexecution by one or more computer processors, implements any of themethods above or elsewhere herein.

Another aspect of the present disclosure provides a system comprisingone or more computer processors and computer memory coupled thereto. Thecomputer memory comprises machine executable code that, upon executionby the one or more computer processors, implements any of the methodsabove or elsewhere herein.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 shows an example of a microfluidic channel structure forpartitioning individual biological particles.

FIG. 2 shows an example of a microfluidic channel structure fordelivering barcode carrying beads to droplets.

FIG. 3 shows an example of a microfluidic channel structure forco-partitioning biological particles and reagents.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.FIG. 7B shows a perspective view of the channel structure of FIG. 7A.

FIG. 8 illustrates an example of a barcode carrying bead.

FIG. 9 shows a diagram of methods for capturing molecules in or around acell bead in a droplet.

FIG. 10 illustrates an exemplary scheme for cell bead generation.

FIG. 11 illustrates an exemplary scheme for cell bead generation orfunctionalization using crosslinks.

FIGS. 12A-B illustrate exemplary scheme for polymerization orcrosslinking of polymer or gel precursors to generate cell beadscomprising attached nucleic acid molecules.

FIG. 13 illustrates an exemplary scheme for cell bead generation and forthe generation of partitions comprising cell beads and barcode beads.

FIG. 14 shows a structure of a micrococcal nuclease (MNase).

FIGS. 15A-C illustrates an example method for inhibiting an engineeredenzyme.

FIG. 16 illustrates an exemplary method for fragmenting and barcodingchromatin fragments, using an engineered nuclease.

FIG. 17 illustrates an exemplary method for inhibiting a nuclease usinga functionalized high molecular weight polymer.

FIG. 18 illustrates an exemplary method for processing a nucleic acidmolecule using an enzyme linked to a high molecular weight polymer.

FIG. 19 illustrates an exemplary method for capturing a nucleic acidmolecule using a functionalized high molecular weight polymer.

FIG. 20 shows an exemplary method for epigenetic profiling of chromatinfrom single cells.

FIG. 21 shows a computer system that is programmed or otherwiseconfigured to implement methods provided herein.

FIG. 22 illustrates another example of a barcode carrying bead.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Where values are described as ranges, it will be understood that suchdisclosure includes the disclosure of all possible sub-ranges withinsuch ranges, as well as specific numerical values that fall within suchranges irrespective of whether a specific numerical value or specificsub-range is expressly stated.

The term “barcode,” as used herein, generally refers to a label, oridentifier, that conveys or is capable of conveying information about ananalyte. A barcode can be part of an analyte. A barcode can beindependent of an analyte. A barcode can be a tag attached to an analyte(e.g., nucleic acid molecule) or a combination of the tag in addition toan endogenous characteristic of the analyte (e.g., size of the analyteor end sequence(s)). A barcode may be unique. Barcodes can have avariety of different formats. For example, barcodes can include:polynucleotide barcodes; random nucleic acid and/or amino acidsequences; and synthetic nucleic acid and/or amino acid sequences. Abarcode can be attached to an analyte in a reversible or irreversiblemanner. A barcode can be added to, for example, a fragment of adeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before,during, and/or after sequencing of the sample. Barcodes can allow foridentification and/or quantification of individual sequencing-reads.

The term “real time,” as used herein, can refer to a response time ofless than about 1 second, a tenth of a second, a hundredth of a second,a millisecond, or less. The response time may be greater than 1 second.In some instances, real time can refer to simultaneous or substantiallysimultaneous processing, detection or identification.

The term “subject,” as used herein, generally refers to an animal, suchas a mammal (e.g., human) or avian (e.g., bird), or other organism, suchas a plant. For example, the subject can be a vertebrate, a mammal, arodent (e.g., a mouse), a primate, a simian or a human. Animals mayinclude, but are not limited to, farm animals, sport animals, and pets.A subject can be a healthy or asymptomatic individual, an individualthat has or is suspected of having a disease (e.g., cancer) or apre-disposition to the disease, and/or an individual that is in need oftherapy or suspected of needing therapy. A subject can be a patient. Asubject can be a microorganism or microbe (e.g., bacteria, fungi,archaea, viruses).

The term “genome,” as used herein, generally refers to genomicinformation from a subject, which may be, for example, at least aportion or an entirety of a subject's hereditary information. A genomecan be encoded either in DNA or in RNA. A genome can comprise codingregions (e.g., that code for proteins) as well as non-coding regions. Agenome can include the sequence of all chromosomes together in anorganism. For example, the human genome ordinarily has a total of 46chromosomes. The sequence of all of these together may constitute ahuman genome.

The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be usedsynonymously. An adaptor or tag can be coupled to a polynucleotidesequence to be “tagged” by any approach, including ligation,hybridization, or other approaches.

The term “sequencing,” as used herein, generally refers to methods andtechnologies for determining the sequence of nucleotide bases in one ormore polynucleotides. The polynucleotides can be, for example, nucleicacid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), including variants or derivatives thereof (e.g., single strandedDNA). Sequencing can be performed by various systems currentlyavailable, such as, without limitation, a sequencing system byIllumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or LifeTechnologies (Ion Torrent®). Alternatively or in addition, sequencingmay be performed using nucleic acid amplification, polymerase chainreaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR),or isothermal amplification. Such systems may provide a plurality of rawgenetic data corresponding to the genetic information of a subject(e.g., human), as generated by the systems from a sample provided by thesubject. In some examples, such systems provide sequencing reads (also“reads” herein). A read may include a string of nucleic acid basescorresponding to a sequence of a nucleic acid molecule that has beensequenced. In some situations, systems and methods provided herein maybe used with proteomic information.

The term “bead,” as used herein, generally refers to a particle. Thebead may be a solid or semi-solid particle. The bead may be a gel bead.The gel bead may include a polymer matrix (e.g., matrix formed bypolymerization or cross-linking). The polymer matrix may include one ormore polymers (e.g., polymers having different functional groups orrepeat units). Polymers in the polymer matrix may be randomly arranged,such as in random copolymers, and/or have ordered structures, such as inblock copolymers. Cross-linking can be via covalent, ionic, orinductive, interactions, or physical entanglement. The bead may be amacromolecule. The bead may be formed of nucleic acid molecules boundtogether. The bead may be formed via covalent or non-covalent assemblyof molecules (e.g., macromolecules), such as monomers or polymers. Suchpolymers or monomers may be natural or synthetic. Such polymers ormonomers may be or include, for example, nucleic acid molecules (e.g.,DNA or RNA). The bead may be formed of a polymeric material. The beadmay be magnetic or non-magnetic. The bead may be rigid. The bead may beflexible and/or compressible. The bead may be disruptable ordissolvable. The bead may be a solid particle (e.g., a metal-basedparticle including but not limited to iron oxide, gold or silver)covered with a coating comprising one or more polymers. Such coating maybe disruptable or dissolvable.

The term “sample,” as used herein, generally refers to a biologicalsample of a subject. The biological sample may comprise any number ofmacromolecules, for example, cellular macromolecules. The sample may bea cell sample. The sample may be a cell line or cell culture sample. Thesample can include one or more cells. The sample can include one or moremicrobes. The biological sample may be a nucleic acid sample or proteinsample. The biological sample may also be a carbohydrate sample or alipid sample. The biological sample may be derived from another sample.The sample may be a tissue sample, such as a biopsy, core biopsy, needleaspirate, or fine needle aspirate. The sample may be a fluid sample,such as a blood sample, urine sample, or saliva sample. The sample maybe a skin sample. The sample may be a cheek swab. The sample may be aplasma or serum sample. The sample may be a cell-free or cell freesample. A cell-free sample may include extracellular polynucleotides.Extracellular polynucleotides may be isolated from a bodily sample thatmay be selected from the group consisting of blood, plasma, serum,urine, saliva, mucosal excretions, sputum, stool and tears.

The terms “biological particle” or “analyte carrier,” as used herein,generally refers to a discrete biological system derived from abiological sample. The biological particle or analyte carrier maycomprise, or carry therein, an analyte (e.g., biological analyte) ofinterest. In some embodiments, the analyte carrier is itself the analyteof itnerset. The biological particle or analyte carrier may be a virus.The biological particle or analyte carrier may be a cell or derivativeof a cell. The biological particle or analyte carrier may be anorganelle. The biological particle or analyte carrier may be a rare cellfrom a population of cells. The biological particle or analyte carriermay be any type of cell, including without limitation prokaryotic cells,eukaryotic cells, bacterial, fungal, plant, mammalian, or other animalcell type, mycoplasmas, normal tissue cells, tumor cells, or any othercell type, whether derived from single cell or multicellular organisms.The biological particle or analyte carrier may be a constituent of acell. The biological particle or analyte carrier may be or may includeDNA, RNA, organelles, proteins, or any combination thereof. Thebiological particle or analyte carrier may be or may include a matrix(e.g., a gel or polymer matrix) comprising a cell or one or moreconstituents from a cell (e.g., cell bead), such as DNA, RNA,organelles, proteins, or any combination thereof, from the cell. Thebiological particle or analyte carrier may be obtained from a tissue ofa subject. The biological particle or analyte carrier may be a hardenedcell. Such hardened cell may or may not include a cell wall or cellmembrane. The biological particle or analyte carrier may include one ormore constituents of a cell, but may not include other constituents ofthe cell. An example of such constituents is a nucleus or an organelle.A cell may be a live cell. The live cell may be capable of beingcultured, for example, being cultured when enclosed in a gel or polymermatrix, or cultured when comprising a gel or polymer matrix. As usedherein, the terms “biological particle” and “analyte carrier” may beused interchangeably.

The term “cell bead,” as used herein, generally refers to a particlethat comprises (e.g., encapsulates, contains, attaches to, immobilizesto, etc.) a biological particle or analyte carrier (e.g., a cell, anucleus, a fixed cell, a cross-linked cell), a virus, components of, ormacromolecular constituents derived from a cell or virus. A cell beadmay comprise a hydrogel, polymeric, or crosslinked material. Forexample, a cell bead may comprise a virus and/or a cell. In some cases,a cell bead comprises a single cell. In some cases, a cell bead maycomprise multiple cells adhered together. A cell bead may include anytype of cell, including without limitation prokaryotic cells, eukaryoticcells, bacterial, fungal, plant, mammalian, or other animal cell types,mycoplasmas, normal tissue cells, tumor cells, a T-cell (e.g., CD4T-cell, CD4 T-cell that comprises a dormant copy of humanimmunodeficiency virus (HIV)), a fixed cell, a cross-linked cell, a rarecell from a population of cells, or any other cell type, whether derivedfrom single cell or multicellular organisms. Furthermore, a cell beadmay comprise a live cell, such as, for example, a cell capable of beingcultured. Moreover, in some examples, a cell bead may comprise aderivative of a cell, such as one or more components of the cell (e.g.,an organelle, a cell protein, a cellular nucleic acid, genomic nucleicacid, messenger ribonucleic acid, a ribosome, a cellular enzyme, etc.).In some examples, a cell bead may comprise material obtained from abiological tissue, such as, for example, obtained from a subject. Insome cases, cells, viruses or macromolecular constituents thereof areencapsulated within a cell bead. Encapsulation can be within a polymeror gel matrix that forms a structural component of the cell bead. Insome cases, a cell bead is generated by fixing a cell in a fixationmedium or by cross-linking elements of the cell, such as the cellmembrane, the cell cytoskeleton, etc. In some cases, beads may or maynot be generated without encapsulation within a larger cell bead.

The term “macromolecular constituent,” as used herein, generally refersto a macromolecule contained within or from a biological particle oranalyte carrier. The macromolecular constituent may comprise a nucleicacid. In some cases, the biological particle or analyte carrier may be amacromolecule. The macromolecular constituent may comprise DNA. Themacromolecular constituent may comprise RNA. The RNA may be coding ornon-coding. The RNA may be messenger RNA (mRNA), ribosomal RNA (rRNA) ortransfer RNA (tRNA), for example. The RNA may be a transcript. The RNAmay be small RNA that are less than 200 nucleic acid bases in length, orlarge RNA that are greater than 200 nucleic acid bases in length. SmallRNAs may include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA(tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolarRNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA(tsRNA) and small rDNA-derived RNA (srRNA). The RNA may bedouble-stranded RNA or single-stranded RNA. The RNA may be circular RNA.The macromolecular constituent may comprise a protein. Themacromolecular constituent may comprise a peptide. The macromolecularconstituent may comprise a polypeptide.

The term “antigen binding fragment,” “epitope binding fragment,” or“antibody fragment,” as used herein, generally refers to a portion of anantibody (e.g., comprising each domain of the light and heavy chainsrespectively) capable of binding the same epitope/antigen as theantibody. In some instances, such a fragment may bind, be configured tobind, or be capable of binding the same epitope or antigen as theantibody to the same extent. In some instances, such a fragment maybind, be configured to bind, or be capable of binding the same epitopeor antigen as the antibody to a lesser extent Although multiple types ofepitope binding fragments are possible, an epitope binding fragmenttypically comprises at least one pair of heavy and light chain variableregions (VH and VL, respectively) held together (e.g., by disulfidebonds) to preserve the antigen binding site and does not contain all ora portion of the Fc region. Epitope binding fragments of an antibody canbe obtained from a given antibody by any suitable technique (e.g.,recombinant DNA technology or enzymatic or chemical cleavage of acomplete antibody), and typically can be screened for specificity in thesame manner in which complete antibodies are screened. In someembodiments, an epitope binding fragment comprises an F(ab′)₂ fragment,Fab′ fragment, Fab fragment, Fd fragment, or Fv fragment. In someembodiments, the term “antibody” includes antibody-derived polypeptides,such as single chain variable fragments (scFv), diabodies or othermultimeric scFvs, heavy chain antibodies, single domain antibodies, orother polypeptides comprising a sufficient portion of an antibody (e.g.,one or more complementarity determining regions (CDRs)) to conferspecific antigen binding ability to the polypeptide.

The term “molecular tag,” as used herein, generally refers to a moleculecapable of binding to a macromolecular constituent. The molecular tagmay bind to the macromolecular constituent with high affinity. Themolecular tag may bind to the macromolecular constituent with highspecificity. The molecular tag may comprise a nucleotide sequence. Themolecular tag may comprise a nucleic acid sequence. The nucleic acidsequence may be at least a portion or an entirety of the molecular tag.The molecular tag may be a nucleic acid molecule or may be part of anucleic acid molecule. The molecular tag may be an oligonucleotide or apolypeptide. The molecular tag may comprise a DNA aptamer. The moleculartag may be or comprise a primer. The molecular tag may be, or comprise,a protein. The molecular tag may comprise a polypeptide. The moleculartag may be a barcode.

The term “partition,” as used herein, generally, refers to a space orvolume that may be suitable to contain one or more species or conductone or more reactions. A partition may be a physical compartment, suchas a droplet or well. The partition may isolate space or volume fromanother space or volume. The droplet may be a first phase (e.g., aqueousphase) in a second phase (e.g., oil) immiscible with the first phase.The droplet may be a first phase in a second phase that does not phaseseparate from the first phase, such as, for example, a capsule orliposome in an aqueous phase. A partition may comprise one or more other(inner) partitions. In some cases, a partition may be a virtualcompartment that can be defined and identified by an index (e.g.,indexed libraries) across multiple and/or remote physical compartments.For example, a physical compartment may comprise a plurality of virtualcompartments.

Cellular Compositions

Cell beads may be used to trap high molecular weight material (e.g.,genomic DNA), while remaining permeable to small molecules and enzymes.Cell beads may be partitioned into partitions (e.g., droplets, wells).Within these partitions, it may be difficult or impossible to temporallyphase a chemical or biochemical reaction through the sequential additionof an activating or inhibitory agent, which may be required to generatea particular product. For example, it may be desirable to release eitheroriginal fragments of genomic material, or copies of those fragments,from a cell bead through the use of an enzyme, such as a nuclease,transposase, polymerase or reverse transcriptase. In some applications,it is important to spatially partition the activity of the enzyme. Forexample, in an application where fragments of genomic material areexcised from the genome using a nuclease, it may be desirable tospatially control the activity of the nuclease so that it may onlyfunction inside the cell bead within a partition where competingenzymatic activities may be acting on molecular targets. Alternatively,it may be desirable to capture target molecules such as poly-A tailedmRNA or ribosomal RNA (rRNA) that diffuse from the cell bead within theGEM. Disclosed herein are methods and compositions for spatiallypartitioning molecules (e.g., RNA, DNA, protein, small molecules, etc)inside a partition (e.g., a droplet).

In an aspect, provided herein are compositions for nucleic acidanalysis. A composition may comprise a partition comprising a cell bead,a barcoded bead, and a polymer. A partition may be a droplet. A cellbead may comprise a cell. A cell bead may comprise a nucleus. A cellbead may comprise a permeabilized nucleus. A polymer may be incapable ofdiffusing into a cell bead. A polymer may be a functionalized polymer(e.g., may comprise one or more functional components). A polymer may bea linear polymer. A polymer may be a branched polymer. A polymer may beattached to a reagent. A reagent may be, for example, an enzyme, anucleic acid, a chemical compound, or a protein. A polymer may befunctionalized with a chemical compound. A chemical compound may be aninhibitor of an enzyme. A chemical compound may be, for example,deoxythymidine 3′,5′-bisphosphate or ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). A polymer may befunctionalized with a charged species. A polymer may comprise anoligonucleotide comprising a capture sequence. A capture sequence may bea polyT sequence, a random sequence, or an rRNA capture sequence. Apartition may comprise a particle (e.g., a magnetic particle).

A partition (e.g., droplet) may comprise an enzyme. An enzyme may be,for example, a nuclease, transposase, polymerase, helicase, reversetranscriptase, ligase, or phosphatase. In some cases, an enzyme is anuclease. A nuclease may be a micrococcal nuclease (MNase) or a DNase.An enzyme may be an engineered enzyme. An engineered enzyme may be anengineered nuclease. An engineered enzyme may comprise a ligand attachedthereto. A ligand may be, for example, biotin. An engineered nucleasemay be modified with a ligand such that, upon binding of aligand-binding protein to the ligand, the enzyme is inactivated.

An enzyme may be attached to a binding moiety. A binding moiety may bean antibody or fragment thereof. An enzyme may be attached to a bindingmoiety directly (e.g., covalently). An enzyme may be attached to abinding moiety indirectly (e.g., non-covalently). An enzyme may beattached to a binding protein (e.g., Protein A, Protein G). An enzymemay be attached to a binding moiety via a binding protein. A bindingmoiety may be capable of binding to a nucleic acid binding protein(e.g., transcription factor, histone, etc.). A partition may compriseone or more cations for activating an enzyme. Examples of cationsinclude magnesium (Mg²⁺) and calcium (Ca²⁺).

FIG. 9 shows examples of compositions for nucleic acid analysis. Forinstance, in some embodiments, partition 901, such as a droplet in anemulsion or a well (e.g., in a microwell array), comprises cell bead 902comprising analyte carrier or biological particle 903 (e.g., a cell ornucleus) and/or nucleic acids derived from a cell 904. In someinstances, cell bead 902 comprises a polymeric or cross-linked matrix(e.g., a hydrogel) that comprises one or more functional moieties orgroups. For example, the cell bead polymeric or cross-linked matrix(“cell bead matrix”) may comprise one or more nucleic acid molecules.The one or more nucleic acid molecules may comprise one or morefunctional sequences (e.g., 908, 909, and/or 910) configured tofacilitate one or more interactions or reactions with a component (e.g.,a cellular nucleic acid) of partition 901. For example, in someinstances, the cell bead matrix comprises a nucleic acid moleculecomprising a poly-T sequence 908, such as the poly-T sequences describedelsewhere herein. A poly-T sequence may be useful in, for example,capturing mRNA from a sample (e.g., a cell) and/or generating cDNA frommRNA. In other embodiments, the cell bead matrix comprises a nucleicacid molecule comprising a capture sequence 909 configured to hybridizeand/or couple to, e.g., a complementary or partially complementarynucleic acid sequence. A capture sequence 909 may be, for example, arandom nucleotide sequence or an rRNA capture sequence. In otherinstances, a capture sequence 909 can be a targeted sequence, such as asequence configured to be complementary to a cellular nucleic acid, suchas a specific mRNA and/or genomic DNA sequence. In still otherembodiments, the cell bead matrix comprises a nucleic acid moleculecomprising a chemical modification 910 (e.g., 5′-adenylation) configuredto enable covalent capture (e.g., ligation) of a second nucleic acidmolecule. The cell bead matrix may also comprise nucleic acid moleculescomprising other functional nucleic acid sequences, such as a barcodesequence, a unique molecular index (UMI) sequence, a sequencing primersequence (or a partial sequencing primer sequence, such as a partial R1and/or R2 sequence), and/or one or more adaptor sequences, such as asequence configured to attach to the flow cell of a sequencer (e.g., P5,P7), etc. These sequences may be, in some instances, in addition to thefunctional sequences described above (e.g., 908, 909, and/or 910).

Partition 901 (e.g., a droplet) may further comprise bead 905. Bead 905may be a bead as described elsewhere herein (see, for example, FIG. 8).Bead 905 may be a gel bead. In some instances, bead 905 comprises one ormore nucleic acid barcode molecules (e.g., is a barcoded bead) asdescribed in greater detail elsewhere herein. Nucleic acid barcodemolecules may be attached (e.g., releasably attached) to bead 905.

Partition 901 may further comprise a polymer 906. Polymer 906 may be alinear polymer. Polymer 906 may be a branched polymer. In someinstances, polymer 906 is a high molecular weight (HMW) polymer. Forexample, polymer 906 may comprise dextran, polyethylene glycol (PEG),polyacrylamide, agarose, alginate, polyvinyl alcohol, PEG-diacrylate,PEG-acrylate/thiol, PEG-azide/alkyne, other acrylates, chitosan,hyaluronic acid, collagen, fibrin, gelatin, elastin, a polyolefin, anolefin copolymers, an acrylics, a vinyl polymer, a polyesters, apolycarbonate, a polyamide, a polyimide, a formaldehyde resin, apolyurethane, an ether polymer, a cellulosic, a thermoplastic elastomer,a thermoplastic polyurethane, or any polymeric precursor (e.g., monomer)thereof. In some instances, polymer 906 is configured such that polymer906 is incapable of diffusing into cell bead 902. In some instances,polymer 906 is a functionalized polymer. A functionalized polymer may beuseful in spatially partitioning reactions within partition 901. Forexample, polymer 906 may be functionalized with, e.g., a protein orpolypeptide, a chemical compound (e.g., a small molecule), or otherfunctionalized group attached thereto. A protein, polypeptide, orchemical compound attached to polymer 906 may be useful in, for example,selectively inhibiting an enzyme (e.g., following release from cell bead902) or otherwise sequestering a species in partition 901. A protein,polypeptide, or chemical compound may be covalently attached to polymer906. A protein, polypeptide, or chemical compound may be non-covalentlyor indirectly attached to polymer 906, e.g., through functional groupsattached to polymer 906. The protein, polypeptide, or chemical compoundattached to polymer 906 may be capable of binding one or more specificligands. For example, in some embodiments, polymer 906 comprisesstreptavidin or a polypeptide capable of binding biotin. In someinstances, polymer 906 comprises a biotin moiety. In some instances, thechemical compound attached to polymer 906 may be an inhibitor of anenzyme. For example, in some embodiments, deoxythymidine3′,5′-bisphosphate is attached to polymer 906, thereby inhibiting, e.g.,a nuclease, such as micrococcal nuclease (MNase). In another example,polymer 906 is functionalized with a chelation agent such as ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) orethylenediaminetetraacetic acid (EDTA), thereby chelating one or morecofactors present in partition 901.

Polymer 906 may comprise one or more nucleic acid molecules. The one ormore nucleic acid molecules may comprise one or more functionalsequences (e.g., the functional sequences 908, 909, and/or 910 describedabove) configured to facilitate one or more interactions or reactionswith a component (e.g., a cellular nucleic acid) of partition 901. Forexample, in some instances, the polymer 906 comprises a nucleic acidmolecule comprising a poly-T sequence 912, such as the poly-T sequencesdescribed elsewhere herein. In other embodiments, polymer 906 comprisesa nucleic acid molecule comprising a capture sequence (similar tocapture sequence 909) configured to hybridize and/or couple to a nucleicacid sequence, e.g., a complementary or partially complementary nucleicacid sequence. A capture sequence may be, for example, a randomnucleotide sequence or an rRNA capture sequence. In other instances, acapture sequence can be a targeted sequence, such as a sequenceconfigured to be complementary to a cellular nucleic acid, such as aspecific mRNA and/or genomic DNA sequence. In still other embodiments,polymer 906 comprises a nucleic acid molecule comprising a chemicalmodification (e.g., 5′-adenylation as depicted in 910) configured toenable covalent capture (e.g., ligation) of a second nucleic acidmolecule. Polymer 906 may also comprise nucleic acid moleculescomprising other functional nucleic acid sequences, such as a barcodesequence, a unique molecular index (UMI) sequence, a sequencing primersequence (or a partial sequencing primer sequence, such as a partial R1and/or R2 sequence), and/or one or more adaptor sequences, such as asequence configured to attach to the flow cell of a sequencer (e.g., P5,P7), etc. These sequences may be, in some instances, in addition to thefunctional sequences described above (e.g., 908, 909, and/or 910).

Polymer 906 may be a charged polymer or comprise one or more chargedfunctional groups. Polymer 906 may be positively charged. Polymer 906may be negatively charged. A charged polymer may be useful in, forexample, capturing analytes (e.g., one or more cellular components) ofopposite charge. For example, polymer 906 may be a positively chargedpolymer 911, thereby capturing negatively charged nucleic acid moleculesreleased from a cell bead.

Partition 901 (e.g., an aqueous droplet) may further comprise a particle907. Particle 907 may be a magnetic particle. Particle 907 may be aparamagnetic particle. Particle 907 may be a bead (e.g., a magneticbead). Particle 907 may be associated with, or otherwise embeddedwithin, e.g., cell bead 902, bead 905 (e.g., a gel bead), and/or polymer906. Particle 907 may be separate from cell bead 902, bead 905 (e.g., agel bead), and polymer 906 (e.g., contained within an aqueous portion ofpartition 901). Particle 907 may be comprised within one or more of cellbead 902, bead 905 (e.g., a gel bead), polymer 906, and/or within anaqueous portion of partition 901.

Particle 907 may comprise one or more nucleic acid molecules. The one ormore nucleic acid molecules may comprise one or more functionalsequences (e.g., the functional sequences 908, 909, and/or 910 describedabove) configured to facilitate one or more interactions or reactionswith a component (e.g., a cellular nucleic acid) of partition 901. Forexample, in some instances, particle 907 comprises a nucleic acidmolecule comprising a poly-T sequence (similar to poly-T sequences 908and 912). In other embodiments, particle 907 comprises a nucleic acidmolecule comprising a capture sequence (similar to capture sequence 909)configured to hybridize and/or couple to, e.g., a complementary orpartially complementary nucleic acid sequence. A capture sequence maybe, for example, a random nucleotide sequence or an rRNA capturesequence. In other instances, a capture sequence can be a targetedsequence, such as a sequence configured to be complementary to acellular nucleic acid, such as a specific mRNA and/or genomic DNAsequence. In still other embodiments particle 907 comprises a nucleicacid molecule comprising a chemical modification (e.g., 5′-adenylationas depicted in 910) configured to enable covalent capture (e.g.,ligation) of a second nucleic acid molecule. Particle 907 may alsocomprise nucleic acid molecules comprising other functional nucleic acidsequences, such as a barcode sequence, a unique molecular index (UMI)sequence, a template switching oligonucleotide (TSO) sequence (such asthose described elsewhere herein), a sequencing primer sequence (e.g., aR1 and/or R2 sequence or a partial sequencing primer sequence, such as apartial R1 and/or R2 sequence), and/or one or more adaptor sequences,such as a sequence configured to attach to the flow cell of a sequencer(e.g., P5, P7), etc. These sequences may be, in some instances, inaddition to the functional sequences described above (e.g., 908, 909,and/or 910).

In some embodiments, the nucleic acid molecules attached to, e.g. cellbead 902 or polymer 906, are single-stranded nucleic acid molecules. Insome embodiments, the nucleic acid molecules attached to, e.g. cell bead902 or polymer 906, are double-stranded nucleic acid molecules. In someembodiments, the nucleic acid molecules attached to, e.g. cell bead 902or polymer 906, are partially double-stranded nucleic acid molecules.

In some cases, the length of a nucleic acid molecule attached to, e.g.cell bead 902 or polymer 906, is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249 or 250 nucleotides or longer.

In some cases, the length of a nucleic acid molecule attached to, e.g.cell bead 902 or polymer 906, is at least 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249 or 250 nucleotides or longer.

In some cases, the length of a nucleic acid molecule attached to, e.g.cell bead 902 or polymer 906, is at most 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,246, 247, 248, 249 or 250 nucleotides.

Generation of Functionalized Polymers

Cell beads (or polymer 906) comprising a nucleic acid molecule attachedthereto can be generated using any suitable method(s) described herein.For a description of cell beads and cell bead generation strategies, seeU.S. Pat. Pub. US 2018/0216162 and PCT Application PCT/US18/54458, filedOct. 4, 2018, both of which are hereby incorporated by reference intheir entirety. For example, in some embodiments, an analyte carrier(e.g., a cell or cell nucleus) is partitioned into a partition (e.g., adroplet in an emulsion) with polymeric or gel precursors and one or morenucleic acid molecules comprising, e.g., one or more functionalsequences, such as the functional sequences described elsewhere herein(e.g., 908, 909, and/or 910). The partition is subjected to conditionssufficient to polymerize or cross-link the polymeric or gel precursorsto generate the cell bead, wherein the cell bead encapsulates theanalyte carrier and the one or more nucleic acid molecules.

In some cases, cell beads (or polymer 906) can be synthesized inone-step procedures, e.g., polymerization and concurrent cross-linkingreactions of multifunctional monomers. In other cases, cell beads can besynthesized in multi-steps procedures, e.g., polymerization of monomersfirst, followed by crosslinking reactions by using, e.g., orthogonal,reactive groups that can respond to different conditions to allowstepwise approaches.

Cell beads (or polymer 906) can be synthesized by techniques that cancreate a crosslinked polymer. In some cases,copolymerization/cross-linking free radical polymerizations can be usedto produce hydrogels by reacting hydrophilic monomers withmultifunctional crosslinking molecules. This can be done by, forexample, linking polymer chains via a chemical reaction(s), usingionizing radiation to generate main-chain free radicals which canrecombine as crosslinking junctions, or physical interactions such asentanglements, electrostatics, and crystallite formation. Types ofpolymerization can include bulk, solution, and suspensionpolymerization.

Suspension polymerization or dispersion polymerization can be employedin water-in-oil or emulsion processes, sometimes called “inversionsuspension.” In some cases, the monomers and initiators can be dispersedin the oil or hydrocarbon phase as a homogenous mixture. In some cases,two types of polymer molecules can be first produced, each having areactive, crosslinking moiety for cross-linking purposes. Then these twotypes of polymer molecules can be enclosed in an emulsion such that thetwo reactive, crosslinking moieties can react and form crosslinksbetween the two types of polymers, thereby completing the synthesis ofthe hydrogel.

In some cases, cell beads (or polymer 906) can be synthesized frommonomers, polymerization initiators, and crosslinking reagents. Afterthe polymerization reactions are complete, the hydrogels formed can beseparated from remaining starting materials, unwanted by-products, etc.The length of the polymer formed can be controlled depending on thedesired properties of the hydrogels.

Types of polymerizations employed to synthesize hydrogels can include,but are not limited to, free radical polymerization, controlled radicalpolymerization, crosslinking polymerization, networks formation ofwater-soluble polymers, and radiation crosslinking polymerization, etc.Polymerization can be initiated by initiators or free-radical generatingcompounds, such as, for example, benzoyl peroxide,2,2-azo-isobutyronitrile (AIBN), and ammonium peroxodisulphate, or byusing UV-, gamma- or electron beam-radiation.

For example, as shown in FIG. 10, cells and polymer or gel precursorsare mixed with an immiscible fluid (e.g., an oil), thereby generating aplurality of aqueous droplets, including droplet 1001 comprising ananalyte carrier, in this instance a cell 1002. Droplet 1001 may alsocomprise a nucleic acid molecule comprising a functional sequence 1005,as described elsewhere herein. Droplet 1001 is subjected to conditionssufficient for polymerization or gelation of the polymer or gelprecursors to generate a cell bead 1003 comprising cell 1002 and nucleicacid molecule 1005. Gelation may comprise any of the gelation mechanismsand polymers described herein. In some instances, cell bead 1003 issubjected to treatment conditions sufficient to lyse cell 1002,releasing components of the cell into the cell bead. In otherembodiments, cell 1002 is lysed in droplet 1001 prior to polymerizationor gelation of the polymer or gel precursors to generate cell bead 1003comprising nucleic acid molecule 1005. In still other embodiments, cell1002 is permeabilized before, during, or after polymerization orgelation of the polymer or gel precursors. Cell beads are collected togenerate a plurality of cell beads 1004. Cell beads may be stored forfurther processing. In some cases, nucleic acid molecule 1005 may beattached to the cell beads subsequent to polymerization or gelation ofthe polymer or gel precursor. For instance, polymer or gel precursorsmay comprise one or more functional groups that facilitate theattachment of nucleic acid molecule 1005 subsequent to polymerization orgelation of the polymer or gel precursors. In other embodiments, thepolymer or gel precursors and/or nucleic acid molecule 1005 comprisefunctional groups, which facilitate the incorporation of nucleic acidmolecule 1005 into the cell bead during polymerization or gelation ofthe polymer or gel precursors.

In some embodiments, the functionalized nucleic acid molecule(s) 1005are entrapped within the cell bead polymeric and/or crosslinked matrix(also referred to herein as a “cell bead matrix”). In other embodiments,the nucleic acid molecule(s) 1005 are functionalized with chemicalgroups (e.g., acrydite, amine, thiol, etc.) such that the nucleic acidmolecule(s) 1005 are incorporated into or otherwise attached to the cellbead matrix. For example, in a cell bead matrix comprisingpolyacrylamide, the nucleic acid molecule 1005 can comprise an acryditemoiety such that, upon polymerization of acrylamide monomers, thefunctionalized nucleic acid molecule(s) 1005 are incorporated into thecell bead matrix. In some embodiments, both the nucleic acid molecule1005 and/or the cell bead matrix comprise one or more functional groupsconfigured to facilitate attachment of the nucleic acid molecule 1005 tothe cell bead matrix. For example, in some embodiments, generation of acell bead comprising a nucleic acid molecule 1005 comprises: (a)providing a plurality of polymer or gel precursors (e.g., in apartition), wherein the polymer or gel precursors comprise a pluralityof first crosslink precursors; (b) providing a plurality offunctionalized nucleic acid molecules comprising a second crosslinkprecursor; and (c) crosslinking the polymer or gel precursors and thenucleic acid molecules via a reaction between a first section of thefirst crosslink precursors and a second section of the second crosslinkprecursors, thereby forming the cell bead comprising the nucleic acidmolecule(s).

In some instances, the functionalized nucleic acid molecules areirreversibly incorporated into the cell bead matrix. In other instances,the functionalized nucleic acid molecules are reversibly incorporatedinto the cell bead matrix. For example, a functionalized nucleic acidmolecule can be functionalized with a labile moiety as describedelsewhere herein (e.g., a disulfide bond) such that the functionalizednucleic acid molecule, or a portion thereof, is configured to bereleased from the cell bead matrix and/or cell bead.

In some embodiments, the cell bead matrix includes one or more of thefollowing; disulfide crosslinked polyacrylamide, agarose, alginate,polyvinyl alcohol, PEG-diacrylate, PEG-acrylate/thiol, PEG-azide/alkyne,other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin,elastin, a polyolefin, an olefin copolymers, an acrylics, a vinylpolymer, a polyesters, a polycarbonate, a polyamide, a polyimide, aformaldehyde resin, a polyurethane, an ether polymer, a cellulosic, athermoplastic elastomer, a thermoplastic polyurethane, or any polymericprecursor (e.g., monomer) thereof. In some embodiments, the cell beadmatrix comprises polyacrylamide (e.g., disulfide crosslinkedpolyacrylamide).

In some embodiments, generation of the cell bead matrix comprises (a)providing a first polymer or gel precursor, wherein the first polymer orgel precursor comprises a plurality of first crosslink precursors, forexample a moiety comprising an azide group; (b) providing a secondpolymer or gel precursor, wherein the second polymer or gel precursorcomprises a plurality of second crosslink precursors, for example amoiety comprising an alkyne group; and (c) crosslinking the firstpolymer and the second polymer via a reaction (e.g., a click-chemistryreaction) between a first section of the first crosslink precursors anda second section of the second crosslink precursors, thereby forming thecell bead.

For example, as shown in FIG. 11, emulsion systems 1100, 1102, and 1104represent different stages through which polymer molecules or gelprecursors are crosslinked to form a cell bead matrix or hydrogel.Emulsion system 1100 can comprise a discrete droplet 1108 (comprising anaqueous phase) immersed in an oil phase 1110. Within the discretedroplet 1108, two polymer molecules 1112 and 1114 and an analyte carrier(e.g., a single analyte carrier, such as a single cell—not shown) can bepartitioned together. In some instances, a functionalized nucleic acidmolecule (not shown, see e.g., 908, 909, and/or 910) is also partitionedwith the polymer molecules or gel precursors and the analyte carrier. Insome embodiments, the nucleic acid molecule further comprises afunctional group (e.g., a click chemistry moiety such as 1118 or 1120)to facilitate attachment to the cell bead matrix. Polymer molecule 1112can comprise a first crosslink precursor comprising a first clickchemistry moiety 1118 and optionally a labile bond 1116 (e.g., achemically, thermally, enzymatically, or photo-labile bond). Polymermolecule 1114 can comprise a second click chemistry moiety 1120. In theoil phase 1111, there can be other reagents, such as reagent 1122 (shownas a copper (II) reagent), which may be utilized to facilitate the clickchemistry reaction between the first click chemistry moiety 1118 and thesecond click chemistry moiety 1120, either by itself or by a derivativethereof. Because the reagent 1122 remains outside of the discretedroplet 1108, generally no click chemistry reaction happens within thediscrete droplet 1108 in the absence of the reagent 1122.

In emulsion system 1102, some of the reagent 1122 can penetrate thediscrete droplet 1108, via, e.g., physical or chemical processes. Insome instances, reagent 1122 becomes or is otherwise processed to becomereagent 1124 (shown as a copper (I) reagent) in the discrete droplet1108. In some instances, conversion into reagent 1124 requiresadditional reagents (not shown, e.g., a reducing agent such as sodiumascorbate). In these embodiments, reagent 1124 can be the reagentrequired to initiate the click chemistry reaction between the firstclick chemistry moiety 1118 and the second click chemistry moiety 1120.Once in the proximity of both the first click chemistry moiety 1118 andthe second click chemistry moiety 1120, the reagent 1124 can initiate aclick chemistry reaction, such as a Cu(I)—Catalyzed Azide-AlkyneCycloaddition (CuAAC), see emulsion system 1104. In embodiments wherethe functionalized nucleic acid molecules comprise a click-chemistrymoiety, the reagent can also catalyze the attachment of nucleic acidmolecules to the cell bead matrix.

As shown in the emulsion system 1104 of FIG. 11, in the presence of thereagent 1124, a crosslink 1126 is formed linking the two polymermolecules 1112 and 1114 together, via the newly formed moiety 1128because of the click chemistry reaction between the first clickchemistry moiety 1118 and the second click chemistry moiety 1120. Ahydrogel comprising the crosslinked polymer molecules 1112 and 1114 canthus be formed, thereby generating the cell bead. Reagents 1122 and/or1124 can be removed from the newly formed hydrogel if desired. In someinstances, the cell bead matrix comprises a labile bond 1116 (e.g., adisulfide bond) configured to release the crosslinks 1126 and/or degradethe hydrogel upon application of a stimulus (e.g., a chemical, thermal,or photo-stimulus). In some instances, the nucleic acid molecules areattached to the hydrogel via a labile bond 1116 configured to releasethe nucleic acid molecules from the cell bead matrix.

In some embodiments, the nucleic acid molecule(s) described herein areattached, entrapped, or otherwise incorporated into the cell bead matrixduring cell bead generation (see, e.g., FIG. 10 and FIG. 11). In otherembodiments, the nucleic acid molecule(s) described herein are attached,entrapped, or otherwise incorporated into the cell bead matrixsubsequent to cell bead generation. For example, in some instances, acell bead can be generated as described elsewhere herein and a nucleicacid molecule can be attached to the cell bead matrix by a chemicalreaction, e.g., between a functional group of the nucleic acidmolecule(s) and a functional group in the cell bead matrix.

FIGS. 12A-B illustrates an example of generating cell beads comprisingfunctionalized molecule(s) attached to a polymer matrix. For instance,as shown in FIG. 12A, a partition 1200 comprising gel or polymerprecursors 1201 attached to a nucleic acid molecule(s) 1202 (e.g., anucleic acid molecule comprising functional sequences such as 908, 909,and/or 910) can be subjected to conditions sufficient to polymerize,gel, or crosslink the precursors 1201, thereby generating a cell bead1210 comprising nucleic acid molecule(s) 1202 attached to the polymermatrix 1203. In some instances, a partition 1220 comprising a firstpolymer or gel precursor 1201 attached to nucleic acid molecule(s) 1202and a second polymer or gel precursor 1204 can be subjected toconditions sufficient to polymerize, gel, or crosslink precursors 1201and 1204, thereby generating a cell bead 1230 comprising nucleic acidmolecule(s) 1202 attached to a polymer 1205 of polymer or gel precursors1201 and 1204. In some instances, polymer or gel precursor 1201 is afirst type of polymer, polymer or gel precursor 1204 is a second type ofpolymer, and polymer 1205 is a copolymer of precursors 1201 and 1204. Inother instances, polymer or gel precursor 1201 is a first type ofpolymer comprising a nucleic acid molecule(s) 1202 and polymer or gelprecursor 1204 is the same type of polymer as 1201 but lacks nucleicacid molecule 1202.

In other embodiments, as shown in FIG. 12B, a partition 1240 is providedcomprising gel or polymer precursors 1201 comprising a first crosslinkprecursor 1206 (e.g., a first click chemistry moiety) and a nucleic acidmolecule(s) 1202 (e.g., a nucleic acid molecule comprising functionalsequences such as 908, 909, and/or 910) comprising a second crosslinkprecursor 1207 (e.g., a second click chemistry moiety), wherein thefirst crosslink precursor 1206 and the second crosslink precursor 1207are configured to form a crosslink 1209 thereby linking the nucleic acidmolecule(s) 1202 with the polymer or gel precursor 1201 or with apolymerized gelled, or otherwise crosslinked matrix of 1201 (e.g.,1211).

In some instances, a partition 1260 is provided comprising (i) a firstpolymer or gel precursor 1201 comprising a first crosslink precursor1206 (e.g., a first click chemistry moiety), (ii) a second polymer orgel precursor 1204, and (iii) a nucleic acid molecule 1202 comprising asecond crosslink precursor 1207 (e.g., a second click chemistry moiety),wherein the first crosslink precursor 1206 and the second crosslinkprecursor 1207 are configured to form a crosslink 1209 thereby linkingthe nucleic acid molecule 1202 with the polymer or gel precursor 1201 orwith a polymerized, gelled, or otherwise crosslinked matrix of 1201 and1212 (e.g., 1213). In some instances, a partition 1260 comprising thefirst polymer or gel precursor 1201 attached to nucleic acid molecule1202 and the second polymer or gel precursor 1212 are subjected toconditions sufficient to polymerize, gel, or crosslink precursors 1201and 1212, thereby generating a cell bead 1270 comprising nucleic acidmolecule(s) 1202 attached to a polymer or gel 1213 of polymer or gelprecursors 1201 and 1212. In some instances, polymer or gel precursor1201 is a first type of polymer, polymer or gel precursor 1212 is asecond type of polymer, and polymer 1213 is a copolymer of precursors1201 and 1212. In other instances, polymer or gel precursor 1201 is afirst type of polymer comprising a nucleic acid molecule 1202 andpolymer or gel precursor 1212 is the same type of polymer as 1201 butlacks the nucleic acid molecule 1202.

In some instances, one or more agents are utilized to catalyze,initiate, or otherwise facilitate the formation of crosslink 1209. Insome instances, the partition 1240 is subjected to conditions sufficientto form a crosslink 1209 between crosslink precursors 1206 and 1209prior to polymerization, gelling, or crosslinking of polymer precursors(e.g., 1201 and/or 1212) to form cell bead 1250 or 1270. In otherinstances, the partition (e.g., 1240 or 1260) is subjected to conditionssufficient to form a crosslink 1209 between crosslink precursors 1206and 1209 concurrently with the polymerization, gelling, or crosslinkingof the polymer or gel precursors (e.g., 1201 and/or 1212). In someembodiments, the partition (e.g., 1240 or 1260) is subjected toconditions sufficient to polymerize, gel, or otherwise crosslink thepolymer or gel precursors (e.g., 1201 and/or 1212) prior to forming acrosslink 1209 between crosslink precursors 1206 and 1209. In someinstances, the nucleic acid molecule 1202 comprises a labile bond 1208configured to release the crosslink 1209 and the nucleic acid molecule1202 upon application of a stimulus (e.g., a chemical, thermal, orphoto-stimulus).

In some instances, a nucleic acid molecule 1202 is attached to the firstpolymer or gel precursor (e.g., 1201), the second polymer or gelprecursors (e.g., 1204 or 1212), or both the first 1201 and the secondpolymer or gel precursors (e.g., 1204 or 1212). Furthermore, in someembodiments, additional polymers or polymer or gel precursors can beadded (e.g., to partition 1200, 1220, 1240, or 1260) to generate aco-polymer or mixed polymer cell bead matrix. Additionally, theconcentration of polymers (e.g., 1201, 1204, and/or 1212) in thepartition (e.g., 1200, 1220, 1240, or 1260) can be controlled togenerate a cell bead comprising a desired concentration of nucleic acidmolecules 1202.

Although FIGS. 12A-B have been generally described above in terms ofattaching, e.g., nucleic acid molecules to cell beads, these and othersuitable methods are equally applicable in generating functionalizedpolymers (such as polymer 906). For example, a plurality of polymerprecursors (e.g., 1201) comprising a nucleic acid molecule (e.g., 1202)can be subjected to conditions sufficient to polymerize, gel, orcrosslink the precursors to generate functionalized polymer 906comprising nucleic acid molecules (e.g., 1202 comprising functionalsequences such as 908, 909, and/or 910). Similarly, although FIGS. 12A-Bhave been generally described above in terms of attaching, e.g., nucleicacid molecules to, e.g., the cell bead 902 or polymer 906, these andother suitable methods are equally applicable in generating cell beadsand polymers comprising other functional molecules such as: chemicalspecies (e.g., biotin, small molecule inhibitors of an enzyme, chelatingagents (e.g., aminopolycarboxylates, such as EDTA and EGTA)), aptamers,proteins or polypeptides (e.g., streptavidin, protein A, protein G,antibodies or antigen binding fragments thereof, nanobodies, etc.),enzymes (e.g., a nuclease, transposase, polymerase, helicase, reversetranscriptase, ligase, phosphatase, etc.), lipids, carbohydrates,polysaccharides, and/or glycoproteins.

In some cases, the polymers (e.g., cell bead 902 and/or polymer 906)disclosed herein can comprise poly(acrylic acid), poly(vinyl alcohol),poly(vinylpyrrolidone), poly(ethylene glycol), polyacrylamide, somepolysaccharides, or any derivatives thereof. These polymers can benon-toxic and they can be used in various pharmaceutical and biomedicalapplications. Thus, in some instances, they may not require theirremoval from the reaction system, thereby eliminating the need for apurification step after the formation of hydrogels.

Polymers (e.g., cell bead 902 and/or polymer 906) can comprise polymermolecules of a particular length or range of lengths. Polymer moleculescan have a length of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,250, 300, 350, 400, 450, 500, 1,000, 2,000, 5,000, 10,000, 20,000,50,000, 100,000, 200,000, 500,000, 1,000,000, 2,000,000, 5,000,000,10,000,000, 20,000,000, 100,000,000, 200,000,000, 500,000,000 or1,000,000,000 backbone atoms or molecules (e.g., carbons). Polymermolecules can have a length of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 1,000, 2,000,5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000,2,000,000, 5,000,000, 10,000,000, 20,000,000, 100,000,000, 200,000,000,500,000,000 or 1,000,000,000 backbone atoms or molecules (e.g.,carbons). Polymer molecules can have a length of at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500,1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000,1,000,000, 2,000,000, 5,000,000, 10,000,000, 20,000,000, 100,000,000,200,000,000, 500,000,000 or 1,000,000,000 monomer units (e.g., vinylmolecules or acrylamide molecules). Polymer molecules can have a lengthof at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250,300, 350, 400, 450, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000,100,000, 200,000, 500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000,20,000,000, 100,000,000, 200,000,000, 500,000,000 or 1,000,000,000monomer units (e.g., vinyl molecules or acrylamide molecules).

Partitioning Cell Beads

Cell beads (optionally comprising nucleic acid molecules comprisingfunctional sequences, e.g., 908, 909, and/or 910) may be partitionedtogether with nucleic acid barcode molecules (optionally attached to abead, e.g., 905) and the nucleic acid molecules of or derived from theanalyte carrier of the cell bead (e.g., mRNA, cDNA, gDNA, etc,) may bebarcoded as described elsewhere herein. In some embodiments, cell beadsare co-partitioned (e.g., in a well or droplet emulsion) with a polymer(e.g., 906), and barcode carrying beads 905 (e.g., gel beads) and thenucleic acid molecules of or derived from the cell bead 904 are barcodedas described elsewhere herein. An overview of an exemplary method forgenerating partitions comprising cell beads and nucleic acid barcodemolecules is schematically depicted in FIG. 13. The method described inFIG. 13 comprises three phases 1310, 1320, and 1330 with each respectivephase comprising: (1) generation of cell beads (1310); (2) cell beadsolvent exchange and optional processing (1320); and (3) co-partitioningof cell beads and barcodes for subsequent tagging (e.g., barcoding) ofone or more constituents of (or derived from) the cell bead (1330).

With continued reference to FIG. 13, phase 1310 comprises providing anoil 1301, polymeric or gel precursors 1302, and analyte carriers 1303(e.g., a cell, a fixed cell, a cross-linked cell, a nucleus, apermeabilized nuclei, etc.) to a microfluidic chip (e.g., 1304) fordroplet generation. Functionalized nucleic acid molecules, such as thosedescribed elsewhere herein, may be further provided to microfluidic chip1304 for co-partitioning. In some instances, the functionalized nucleicacid molecules (e.g., 908, 909, and/or 910) are provided with orotherwise attached to the polymeric or gel precursors 1302. In othercases, the functionalized nucleic acid molecules are provided with theanalyte carriers 1303. In some instances, the microfluidic chip 1304comprises a plurality of microfluidic channels (see e.g., FIGS. 1-7)connected to a plurality of reservoirs comprising the oil 1301,polymeric or gel precursors 1302, and analyte carriers (e.g., cells)1303. Microfluidic chip 1304 may also comprise one or more additionalchannels and/or reservoirs comprising one or more additional reagents(such as the functional nucleic acid molecules described herein).Polymeric or gel precursors 1302 and analyte carriers 1303 (and in somecases, functional nucleic acid molecules) are flowed (e.g., via theaction of an applied force, such as negative pressure via a vacuum orpositive pressure via a pump) from their reservoirs through theplurality of microfluidic channels to a first channel junction andcombine to form an aqueous stream. This aqueous stream is then flowed toa second channel junction, in which oil 1301 is provided. The aqueousstream provided from the first channel junction is immiscible with theoil 1301 resulting in the generation of a suspension of aqueous droplets1305 in the oil, which then flow to a reservoir for collection. Flow canbe controlled within the microfluidic chip 1304 via any suitable method,including the use of one or more flow regulators in a channel or variouschannels, dimensioning of microfluidic channels, etc., as describedelsewhere herein. As shown in FIG. 13, the product comprises droplets1305 comprising an analyte carrier 1303, the polymeric or gel precursors1302, and in some cases, nucleic acid molecules comprising functionalsequences (such as 908, 909, and/or 910). In some cases, at least someof the droplets of droplets 1305 comprise a single analyte carrier(e.g., a single cell or single nucleus).

In some embodiments, the droplets 1305 are subjected to conditionssufficient to lyse the analyte carriers (e.g., cells or nuclei)comprised therein, releasing cellular macromolecular constituents intothe droplets 1305. The macromolecular constituents (e.g., nucleic acids,proteins, etc.) may additionally be subjected to one or more reactionsfor processing as described elsewhere herein. In other embodiments, thedroplets 1305 are subjected to conditions sufficient to permeabilize thecells (or nuclei) thereby facilitating access to one or moremacromolecular constituents of the cell (or nucleus) for furtherprocessing. In still other cases, the analyte carriers present in thedroplets 1305 are not lysed or permeabilized.

Continuing with FIG. 13, the droplets 1305 comprising analyte carriersare then subjected to conditions suitable to polymerize or gel thepolymeric or gel precursors 1302 in the droplets 1305, to generate cellbeads 1306. As the resulting cell beads 1306 are suspended in oil, insome embodiments, phase 1320 is initiated which comprises a solventexchange configured to resuspend the cell beads 1306 in an aqueous phase1311.

In some embodiments, the resuspended aqueous cell beads 1311 areoptionally processed to, e.g., prepare the cell beads for analysis ofone or more cellular components. For example, cell beads 1311 can besubjected conditions suitable to lyse or permeabilize analyte carriers(e.g., cells or nuclei) in the cell beads 1313, thereby releasing orotherwise allowing access to one or more cellular constituents (e.g.,nucleic acids, such as mRNA and gDNA, proteins, etc.). Separately orcontemporaneously from cell lysis, cell beads (e.g., 1311 or 1313) arealso subjected to conditions sufficient to denature nucleic acidsderived from the cells (e.g., gDNA) associated with the cell beads(e.g., using NaOH). The polymeric matrix of the cell beads (e.g., 1311or 1313) may effectively hinder or prohibit diffusion of largermolecules, such as nucleic acids and/or proteins, from the cell beads,but may be sufficiently porous to facilitate diffusion of denaturationor other agents into the cell bead matrix to contact nucleic acids andother cellular components within the cell beads. In some cases, the cellbeads (1311 or 1313) can be subjected to conditions suitable forperforming one or more reactions on nucleic acids or other analytesderived from the cells associated with the cell beads (1311 or 1313).For example, antibodies or a nuclease (e.g., 1603, 1703 as describedelsewhere herein) may be washed into and/or out of the resuspended cellbeads (1311 or 1313). Additionally, in embodiments where functionalnucleic acid molecules are attached or otherwise incorporated into thecell beads subsequent to cell bead generation, functional nucleic acidmolecules can be provided and one or more reactions performed on thecell bead (1311 or 1313) to attach or otherwise incorporate thefunctional nucleic acid molecules into the cell beads (e.g., throughfunctional groups on the functional nucleic acid molecule(s), cell beadmatrix, or both). After optional processing, the cell beads comprisingcellular constituents can be collected 1314 and stored prior toinitiation of phase 1330.

Continuing with FIG. 13, after phase 1320, cell beads 1314 can beanalyzed by, e.g., partitioning cell beads and nucleic acid barcodemolecules into partitions (e.g., droplets, microwells) for analysis ofcellular components (e.g., nucleic acid molecules). For example, inphase 1330, partitions (e.g., droplets) comprising cell beads 1314 andbeads (e.g., a gel bead) comprising nucleic acid barcode molecules 1322(“barcode beads”) are generated such that at least some dropletscomprise a cell bead and a barcode bead (e.g., a single cell bead and asingle barcode bead). In some instances, the partition further comprisesa functionalized polymer (e.g., 906). For example, in some embodiments,an oil 1321, the cell beads 1314, barcode beads 1322 each comprising abarcode sequence (e.g., each bead comprising a unique barcode sequence),and a functionalized polymer (e.g., 906) are provided to a microfluidicchip 1323. Exemplary microfluidic chip architecture is shown in e.g.,FIGS. 1-7, but any suitable microfluidic chip or microwell array canalso be utilized with the compositions, methods, and systems disclosedherein. The microfluidic chip 1323 comprises a plurality of reservoirscomprising the oil 1321, cell beads 1314, barcode beads 1322 (e.g., gelbeads), and the high molecular weight functionalized polymer. The chipcan also include additional reservoirs that may be used to supplyadditional reagents (e.g., reagents for nucleic acid amplification,reagents that can degrade or dissolve cell beads and/or gel beads,reagents that degrade linkages between barcode beads/cellbeads/polymers, reagents for cell lysis, etc.). Cell beads 1314 andbarcode beads 1322 are flowed (e.g., via the action of an applied force,such as negative pressure via a vacuum or positive pressure via a pump)from their reservoirs to, e.g., a first channel junction and form anaqueous mixture.

Alternatively, cell beads, barcode beads (e.g., gel beads), and/orpolymers can be mixed before introduction into the microfluidic chip. Inthis case, a single reservoir of the microfluidic chip (e.g., 1323)comprises a mixture of cell beads, barcode beads (e.g., gel beads),and/or polymers. The ratio of cell beads to barcode beads in the mixturecan be varied to alter the number of droplets generated that comprise asingle cell bead and a single barcode bead. The mixture of cell beadsand barcode beads may be flowed (e.g., via the action of an appliedforce, such as negative pressure via a vacuum or positive pressure via apump) from the reservoir to a first channel junction, in some casestogether with materials from additional reservoirs.

In some embodiments, the aqueous mixture comprising cell beads 1314,barcode beads 1322, and in some cases additional reagents is then flowedto a second channel junction, to which oil 1321 is provided. The aqueousmixture provided from the first channel junction is immiscible with theoil 1321 resulting in the generation of a suspension of aqueous droplets1325 in the oil which then flow to a reservoir for collection. Flow canbe controlled within the microfluidic chip 1323 via any suitablestrategy, including the use of one or more flow regulators in a channelor that connect channels, use of various channels, dimensioning ofchannels, etc. In some cases, at least some droplets of droplets 1325comprise a single cell bead and a single barcode bead (e.g., a singlegel bead).

Where reagents that degrade or dissolve the cell beads 1314, barcodebeads 1322 (e.g., gel beads) and/or linkages between barcodes andbarcode beads 1322 (or nucleic acid molecules attached to afunctionalized polymer) are present in droplets, these reagents canrelease the nucleic acids trapped in the cell beads 1313, release thebarcodes from the barcode beads 1322, and/or release functionalized acidmolecules from the cell bead matrix or polymer. The nucleic acid barcodemolecules can interact with the released cellular components (e.g.,cellular nucleic acids) and generate barcoded nucleic acid molecules fornucleic acid sequencing as described elsewhere herein. In embodimentswhere the barcode bead (e.g., gel bead) is degraded or nucleic acidbarcode molecules are releasably attached to the barcode bead (e.g., gelbead), the barcoded cellular components (e.g., barcoded cDNA or gDNAfragments) are not attached to the bead. Where a given droplet comprisesa cell bead (e.g., a single cell bead) and a barcode bead (e.g., asingle barcode bead) comprising nucleic acid barcode moleculescomprising a common barcode sequence, the barcoded cellular components(or derivatives thereof) can be associated with the analyte carrier(e.g., a cell or other biological sample, such as a bacterium or virus)of the given cell bead via the common barcode sequence.

Cellular Processing

In some aspects, disclosed herein are methods for processing andanalysis of nucleic acids from one or more cells. The described methodsmay be used in conjunction with one or more compositions disclosedherein. For example, partitions, cell beads, barcode beads, and/orpolymers may be used in methods of nucleic acid analysis.

First, a partition may be generated comprising a cell bead comprising anucleic acid, a nucleic acid barcode molecule, and a polymer. Next, thenucleic acid and the nucleic acid barcode molecule may be used toperform one or more reactions in the partition. A partition may be adroplet. A partition may be a well. A reaction may be performed outsidethe cell bead. A reaction may comprise nucleic acid extension. Areaction may comprise nucleic acid amplification (e.g., PCR, linearamplification. etc.). A reaction may comprise generating a barcodednucleic acid from the nucleic acid and the nucleic acid barcode.

A cell bead may comprise a nucleus. A cell bead may comprise apermeabilized nucleus. A cell bead may comprise a nucleic acid (e.g.DNA, RNA, etc) derived from a cell and/or a nucleus. A nucleic acidbarcode molecule may be attached to a barcoded bead. A nucleic acidbarcode molecule may be releasably attached to a barcoded bead.

A polymer may be incapable of diffusing into a cell bead. A polymer(e.g., polymer 906) may be a functionalized polymer (e.g., may compriseone or more additional reagents). A polymer may be a linear polymer. Apolymer may be a branched polymer. A polymer may be attached to areagent. A reagent may be, for example, an enzyme, a nucleic acid, achemical compound, or a protein. A polymer may be functionalized with achemical compound. A chemical compound may be an inhibitor of an enzyme.A chemical compound may be, for example, deoxythymidine3′,5′-bisphosphate or ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). A polymer may befunctionalized with a charged species. A polymer may comprise anoligonucleotide comprising a capture sequence. A capture sequence may bea poly-T sequence, a random sequence, or an rRNA capture sequence.

A partition (e.g., droplet) may comprise an enzyme. An enzyme may be,for example, a nuclease, transposase, polymerase, helicase, reversetranscriptase, ligase, or phosphatase. In some cases, an enzyme is anuclease. A nuclease may be a micrococcal nuclease (MNase) or a DNase.An enzyme may be an engineered enzyme. An engineered enzyme may be anengineered nuclease. An engineered enzyme may comprise a ligand attachedthereto. A ligand may be, for example, biotin. An engineered nucleasemay be modified with a ligand such that, upon binding of aligand-binding protein to the ligand, the enzyme is inactivated.

An enzyme may be attached to a binding moiety. A binding moiety may bean antibody or fragment thereof. An enzyme may be attached to a bindingmoiety directly (e.g., covalently). An enzyme may be attached to abinding moiety indirectly (e.g., non-covalently). A nuclease may beattached to a binding protein (e.g., Protein A, Protein G). An enzymemay be attached to a binding moiety via a binding protein. A bindingmoiety may be capable of binding to a nucleic acid binding protein(e.g., transcription factor, histone, etc.). A partition may compriseone or more cations for activating an enzyme. Examples of cationsinclude magnesium (Mg²⁺) and calcium (Ca²⁺).

Compositions and methods described herein may comprise one or moreenzymes. Enzymes may be useful in, for example, performing one or morereactions with nucleic acid(s) in or derived from a cell. An enzyme foruse in the disclosed methods and compositions may be any suitable enzymecapable of modifying or processing a nucleic acid. An enzyme may be atransposase. An enzyme may be a polymerase. An enzyme may be a helicase.An enzyme may be a reverse transcriptase. An enzyme may be a ligase. Anenzyme may be a phosphatase. An enzyme may be a nuclease. A nuclease maybe a DNase. A nuclease may be a micrococcal nuclease (MNase).

An enzyme may be an engineered enzyme. An engineered enzyme may be anenzyme that has been modified, for example, via protein engineering orchemical modification. An enzyme may be modified via the addition of oneor more chemical moieties (e.g., ligands, aptamers, etc.), therebygenerating an engineered enzyme. An engineered enzyme may be used in thedisclosed methods for performing one or more reactions on a nucleicacid. An engineered enzyme may be engineered such that, e.g., the enzymeis active inside a cell or cell bead and inactive/less active outside acell or cell bead. For example, an enzyme may be engineered to comprisea binding moiety capable of binding to or interacting with a bindingpartner such that when the binding partner is bound to the bindingmoiety, the enzyme has reduced enzymatic activity, is functionallyinactivated, and/or is otherwise sequestered from additional substrates.For example, an engineered enzyme may comprise a binding moiety such asa ligand, chemical compound, chemical group (e.g., alkyne, azide, etc.),small molecule, carbohydrate, aptamer, and/or polypeptide (such as anepitope tag). For example, in some embodiments, the engineered enzymecomprises a biotin moiety, wherein upon interaction of the biotin withits binding partner (e.g., streptavidin), the engineered enzyme isfunctionally inactivated or otherwise sequestered from further enzymaticreactions. An engineered enzyme may comprise a binding moiety within,near, or otherwise adjacent to the active site of the enzyme, such thatbinding of the binding partner thereby inhibits the enzyme (e.g.,through steric effects, reversible/irreversible inhibition, substratecompetition, etc.). The active site surface of an enzyme may bepredicted from protein structure and/or homology and a suitablemodification (e.g., a binding moiety) may be selected to generate theengineered enzyme. For example, FIG. 14 shows a 3-D ribbon structure ofa MNase, highlighting the active site (predicted DNA binding pocket),Ca²⁺ ion (MNase is strictly dependent on calcium for activity), and theDNA binding surface available for chemical modification (e.g., bindingmoiety attachment) and/or antibody or aptamer binding.

For example, FIGS. 15A-15C illustrate an exemplary engineered nucleasecomprising a binding moiety. FIG. 15A shows an enzyme 1520, capable offragmenting nucleic acid 1510 into fragments 1511 within the active siteof the enzyme. FIG. 15B shows an engineered nuclease 1530 comprising abinding moiety 1531 (e.g., a biotin molecule). In the absence of abinding moiety binding partner 1540 (e.g., streptavidin), engineerednuclease 1530 is capable of fragmenting nucleic acid 1510 into fragments1511. FIG. 15C shows engineered nuclease 1530 comprising binding moiety1531 (e.g., a biotin molecule) bound to binding moiety binding partner1540 (e.g., streptavidin). With binding moiety binding partner 1540bound to binding moiety 1531, the enzymatic activity of engineerednuclease 1530 is reduced and/or inhibited, thereby reducing/inhibitingfragmentation of nucleic acid 1510.

An engineered enzyme (e.g., 1530) may be attached to a binding moiety(e.g., 1531). An engineered enzyme may be directly or indirectlyattached to a binding moiety. An engineered enzyme may be directlyattached to a binding moiety via a linker. An engineered enzyme may beindirectly attached to a binding moiety via a binding protein. A bindingmoiety may be an antibody or fragment thereof (e.g., a nanobody, anscFv, etc.). An engineered enzyme may be attached to a binding proteincapable of binding a binding moiety. For example, an enzyme may beattached to Protein A or Protein G. A binding protein may bind to abinding moiety, thereby indirectly attaching an enzyme to the bindingmoiety. A binding moiety may be capable of binding to a nucleic acidbinding protein. For example, a binding moiety may be an antibody orantibody fragment capable of binding to a specific nucleic acid bindingprotein, thereby directing an enzyme to a specific region of a nucleicacid.

FIG. 16 shows an example cellular processing method comprising nucleicacid fragmentation and barcoding. In operation 1610, cellular chromatincomprising nucleosomes 1601 (and/or DNA-binding proteins) and genomicDNA 1602 are provided (e.g., from a cell or nucleus). In operation 1620,engineered nucleases 1603 (e.g., an engineered MNase) are provided, eachcomprising a binding moiety within/adjacent to the active site. Inoperation 1630, cations calcium (Ca²⁺) and/or magnesium (Mg²⁺) areadded, thereby activating engineered nucleases 1603 and fragmenting DNA1602 to generate nucleic acid fragments 1604. The cations may be addedby, for example, co-partitioning the chromatin, engineered nucleases1603, and cations into a partition. In operation 1640, binding moietybinding proteins 1605 and barcoded adaptors 1606 (optionally attached toa bead) are provided. As such, in some instances, operations 1610 and1620 may be performed outside a partition, while operations 1630 and1640 may be performed inside one or more partitions (e.g., a droplet orwell). Binding moiety binding proteins 1605 bind to and inhibitengineered nucleases 1607. Ligases (not shown in FIG. 16) are used toattach barcoded adaptors 1606 to the nucleic acid fragments 1604,thereby generating barcoded nucleic acid fragments 1608. Binding proteinbound to engineered nuclease 1607 prevents the engineered nuclease fromdigesting the barcoded nucleic acid fragments 1608. Binding moietybinding proteins 1605 may be attached to a functionalized polymer (e.g.,906), as described elsewhere herein. Although shown as a Y-adapter in1606, any suitable nucleic acid barcode molecule, such as thosedescribed herein, are contemplated.

FIG. 17 shows an example of inhibition of an engineered nuclease using afunctionalized polymer (e.g., 906). Partition 1700 (e.g., an aqueousdroplet in an emulsion or a well of a microwell array) comprises aporous cell bead 1710 with a porosity that allows some molecules (e.g.,1703) to freely diffuse through the cell bead boundary 1720 into theaqueous environment of the partition 1730 while entrapping othermolecules (e.g., 1707) within the cell bead matrix 1710. The size ofmolecules entrapped within the cell bead matrix may be dependent on thecharacteristics of the cell bead (e.g., polymer makeup, porosity, etc.).Within the cell bead matrix 1710, nucleases 1703 (e.g., MNase or atransposase, such as an adapter-loaded transposase) are capable offragmenting chromatin to generate nucleic acid fragments 1702 (e.g.,attached to nucleosomes 1701 or free nucleic acid moleculesrepresentative of areas of accessible chromatin, such as in ATAC-seq).The nuclease 1703 may be an engineered nuclease and comprise a bindingmoiety (e.g., biotin, polypeptide, aptamer, etc.) as described herein.When engineered nuclease 1703 diffuses through the cell bead boundary1720 it may attach to a functionalized polymer comprising a bindingmoiety binding partner 1705 (e.g., streptavidin), thereby inhibiting theenzymatic activity and/or sequestering the enzyme from reactants. Insome instances, when nuclease 1703 diffuses through the cell beadboundary 1720, it may attach to a functionalized polymer comprising aninhibitor 1706 (e.g., a small molecule inhibitor), thereby inhibitingthe enzymatic activity and/or sequestering the enzyme from reactants. Insome embodiments, when nuclease 1703 diffuses through the cell beadboundary 1720, the nuclease 1703 is inactivated and/or sequestered by afunctionalized polymer comprising a molecule specific for nuclease 1703(e.g., an antibody, aptamer, or other binding moiety that when bound tonuclease 1703 occludes the active site from interacting withsubstrate—see, e.g., FIG. 14). In still other instances, when nuclease1703 diffuses through the cell bead boundary 1720, the nuclease 1703 isinactivated by a functionalized polymer comprising a small moleculeinhibitor (e.g., deoxythimidine 3′,5′-bisphosphate, EDTA, EGTA, etc.).Polymer complexes (e.g., 1705 and 1706) are incapable of diffusing intocell bead 1710. A ligase or polymerase (not shown in FIG. 17) can thenattach nucleic acid barcode molecules 1704 (which are permeable to cellbead 1710 and may optionally be attached to a bead, e.g., 905) tonucleic acid fragments 1702. Inhibition of the nucleases 1703 by bindingto the functionalized polymer (e.g., 1705 and/or 1706) prevents removalof the nucleic acid barcode molecules from the nucleic acid fragments.Although shown as a Y-adapter in 1704, any suitable nucleic acid barcodemolecule, such as those described herein, are contemplated.

FIG. 18 shows an example of a polymer 1804 functionalized with an enzyme1805 (e.g., a nuclease, transposase, polymerase, helicase, reversetranscriptase, ligase, phosphatase, or any other suitable enzyme thatmodifies DNA or RNA) for use in spatially controlling reactions such asnucleic acid barcoding. Partition 1800 (e.g., an aqueous droplet in anemulsion or a well of a microwell array) comprises a porous cell bead1810 with a cell bead boundary 1820. Within the cell bead 1810,nucleases (e.g., MNase or a transposase, such as an adapter-loadedtransposase) are capable of fragmenting chromatin to generate nucleicacid fragments 1802 (e.g., attached to nucleosomes 1801 or free nucleicacid molecules representative of areas of accessible chromatin, such asin ATAC-seq). Partition 1800 also comprises nucleic acid barcodemolecules 1803 (which are permeable to cell bead 1810 and may optionallybe delivered to partition 1800 on a bead, e.g., 905). Although shown asa Y-adapter in 1803, any suitable nucleic acid barcode molecule, such asthose described herein. are contemplated. Outside the cell bead 1810,functionalized polymer 1804 may comprise an enzyme 1405 (e.g., aligase). Polymer 1804, attached to enzyme 1805, is incapable ofdiffusing into through the semi-permeable cell bead boundary 1810 intocell bead 1820. Nucleic acid fragments 1802, nucleosomes 1801, and/ornucleic acid barcode molecules 1803 may diffuse out of the porous cellbead 1810 and into the aqueous partition environment 1830. Enzyme 1805(e.g., a ligase or polymerase) may be used to attach nucleic acidbarcode molecules 1803 to the nucleic acid fragments 1802 outside thecell bead 1810.

FIG. 19 shows an example of a polymer 1902 functionalized with a nucleicacid molecule 1903 comprising capture sequences configured to capturenucleic acids from a cellular sample. Partition 1900 (e.g., an aqueousdroplet in an emulsion or a well of a microwell array) comprises aporous cell bead 1910 with a cell bead boundary 1910. Within the cellbead 1910 are cellular nucleic acid molecules 1901 (e.g., mRNAmolecules, rRNA molecules, gDNA fragments, etc.). Nucleic acid molecules1901 may be released from a cell or nucleus within the cell bead 1910.Outside the cell bead, functionalized polymer 1902 may comprise anucleic acid molecule 1903 comprising a capture sequences (e.g.,targeted capture sequences, rRNA capture sequences, random N-mersequences, poly-T sequences, etc.) and optionally an affinity handle1904 (e.g., streptavidin, GST, His-tag, an antibody or portion thereof,etc.) configured to facilitate isolation, removal, purification, etc. ofthe polymer-bound molecules. Nucleic acid molecule 1903 may furthercomprise functional sequences such as barcode sequences, UMIs,sequencing adaptors, or any other functional sequence. Nucleic acidmolecules 1901 may diffuse out of the porous cell bead 1901 and attach(e.g., hybridize or be ligated) to capture sequences in nucleic acidmolecule 1903 to form complexes 1905. Nucleic acid molecules 1901 boundin complexes 1905 may be further processed in partition 1900 or in bulk(e.g., to generate cDNA from mRNA molecules). Complexes 1905 may bepurified from partition 1900, for example, by use of affinity handle1904, thereby isolating nucleic acid molecules (e.g., RNA, cDNAmolecules, etc.).

Epigenetic Profiling

In some aspects, disclosed herein are methods for generating a barcodednucleic acid. A barcoded nucleic acid may be useful in, for example,epigenetic profiling of one or more cells. First a cell bead may begenerated comprising a permeabilized nucleus and a nuclease. In somecases, a cell bead may further comprise a particle (e.g., a magneticparticle). Next, a cell bead, nucleic acid barcode molecule, and apolymer may be co-partitioned into a partition. A polymer may befunctionalized with an inhibitor of the nuclease. Next, the nuclease maybe used to fragment a nucleic acid from the nucleus, thereby generatinga nucleic acid fragment. Next, the nucleic acid fragment and thenuclease may be released from the cell bead into the partition. Thenuclease may come in contact with the inhibitor attached to the polymer,thereby inhibiting the nuclease. Finally, the nucleic acid fragment andthe nucleic acid barcode molecule may be used to generate a barcodednucleic acid. For example, a ligase enzyme may be used to attach thenucleic acid barcode molecule to the nucleic acid fragment, or aderivative thereof (e.g., a complement thereof, a nucleic acid extensionproduct thereof, etc.).

A nuclease may be attached to a binding moiety. A binding moiety may bean antibody or fragment thereof. A nuclease may be attached to a bindingmoiety directly (e.g., covalently). A nuclease may be attached to abinding moiety indirectly (e.g., non-covalently). A nuclease may beattached to a binding protein (e.g., Protein A, Protein G). A nucleasemay be attached to a binding moiety via a binding protein. A bindingprotein may localize a nuclease within a cell bead based on the bindingproperties of the binding protein. In one example, a binding protein isan antibody or fragment thereof with specificity for a nucleic acidbinding protein. In this example, a nuclease may be attached to theantibody and therefore localized to the nucleic acid binding protein ina nucleus. A nuclease may be used to fragment a nucleic acid. Alocalized nuclease may fragment nucleic acid only in a given region. Forexample, a nuclease may be localized to the region of a histone and usedto fragment DNA in the region of the histone, generating DNA fragmentscorresponding to the region of DNA around the histone.

In some cases, a barcoded nucleic acid may be released from a partitionfor further processing. A barcoded nucleic acid may be subjected tonucleic acid sequencing, thereby generating sequences corresponding to anucleic acid from a cell or nucleus. For example, a barcoded nucleicacid may generate sequences from genomic DNA of a nucleus correspondingto a given region. The region may be determined based on localization ofa nuclease within a nucleus. Sequences may be analyzed to generate agenetic and/or epigenetic profile of one or more single cells.

FIG. 20 shows an example method for epigenetic profiling of chromatin.In operation 2000, nuclei from a plurality of cells may be obtained andpermeabilized. Each nucleus may be captured in a cell bead, as describedherein (see, e.g., FIG. 13). In some cases, a cell bead may furthercomprise a particle, such as a magnetic particle. In operation 2010,cell beads may be placed in an aqueous solution comprising a nuclease(e.g., DNase, MNase, etc.) attached to a binding moiety (e.g., anantibody or fragment thereof). A binding moiety may be capable ofbinding to a histone or other nucleic acid binding protein, such as atranscription factor. A nuclease may be attached to a binding moietydirectly (e.g., via covalent linkage). A nuclease may be attached to abinding moiety indirectly. A nuclease may be attached (e.g., covalentlylinked) to a binding protein capable of binding to a binding moiety(e.g., protein A, protein G, etc.). The binding moiety attached to thenuclease (the “tethered nuclease”) may be brought in contact with thechromatin within the nuclei. The binding moiety may bind to a componentof the chromatin (histone, transcription factor, etc.). In some cases,the aqueous solution may not comprise the necessary reagents fornuclease activity (e.g., cations such as Mg²⁺ and Ca²⁺), such that thenuclease is bound to the chromatin in the cell bead in an inactivestate. In some cases, any nuclease which is not bound to the chromatinmay be removed, e.g., by washing the cell beads.

In operation 2020, cell beads may be partitioned into droplets togetherwith a plurality of barcode beads (e.g., gel beads), such that a givendroplet 2001 contains a single cell bead and a single barcoded bead.Additionally, a high molecular weight, functionalized polymer (e.g.,906) may be partitioned into the droplet with the cell bead and barcodedbead. The functionalized polymer may comprise a protein, chemicalcompound, or other means for inactivating the nuclease. For example, thefunctionalized polymer may comprise a chemical compound capable ofinhibiting the nuclease. The functionalized polymer may comprise abinding moiety binding partner (e.g., streptavidin) capable of bindingto a binding partner (e.g., biotin) attached to the nuclease. Thefunctionalized polymer may comprise a chelation agent, such as EGTA orEDTA, capable of chelating one or more cofactors necessary for enzymaticactivity of the nuclease.

The functionalized polymer may comprise nucleic acid moleculescomprising one or more functional sequences (e.g., the functionalsequences 908, 909, and/or 910 described elsewhere herein) configured tofacilitate one or more interactions or reactions with a component (e.g.,a cellular nucleic acid) from a cell bead. The functionalized polymermay also comprise nucleic acid molecules comprising other functionalsequences, such as barcode sequences, a unique molecular index (UMI)sequence, a sequencing primer sequence (or a partial sequencing primersequence, such as a partial R1 and/or R2 sequence), and/or one or moreadaptor sequences, such as a sequence configured to attach to the flowcell of a sequencer (e.g., P5, P7), etc. Droplet 2001 may also comprisethe necessary reagents for nuclease activity (e.g., cations such as Mg²⁺and Ca²⁺), thereby activating the nuclease. The nuclease may fragmentnucleic acid within the chromatin of the nucleus, thereby releasingnucleic acid fragments and the nuclease from the cell bead into thesurrounding droplet. The functionalized polymer may inactivate thenuclease, preventing further DNA fragmentation.

As depicted in FIG. 20, the droplet may also comprise a ligase enzymeand nucleic acid barcode molecules attached to the barcode bead. Theligase enzyme may be used to attach the nucleic acid barcode moleculesto the nucleic acid fragments, thereby barcoding the nucleic acidmolecules from the nucleus. In operation 2030, barcoded molecules maythen be released from the droplets, and cell beads removed, for example,via centrifugation or magnetic pull down. In operation 2040, Barcodednucleic acid molecules may be subjected to amplification (e.g., PCRamplification), purification, and/or nucleic acid sequencing. Nucleicacid sequencing results may be used to identify an epigenetic profile ofeach single cell from the original plurality of cells.

Systems and Methods for Sample Compartmentalization

In an aspect, the systems and methods described herein provide for thecompartmentalization, depositing, or partitioning of one or moreparticles (e.g., analyte carriers, macromolecular constituents ofanalyte carriers, beads, reagents, etc.) into discrete compartments orpartitions (referred to interchangeably herein as partitions), whereeach partition maintains separation of its own contents from thecontents of other partitions. The partition can be a droplet in anemulsion. A partition may comprise one or more other partitions.

A partition may include one or more particles. A partition may includeone or more types of particles. For example, a partition of the presentdisclosure may comprise one or more biological particles (or analytecarriers) and/or macromolecular constituents thereof. A partition maycomprise one or more gel beads. A partition may comprise one or morecell beads. A partition may include a single gel bead, a single cellbead, or both a single cell bead and single gel bead. A partition mayinclude one or more reagents. Alternatively, a partition may beunoccupied. For example, a partition may not comprise a bead. A cellbead can be an analyte carrier and/or one or more of its macromolecularconstituents encased inside of a gel or polymer matrix, such as viapolymerization of a droplet containing the analyte carrier andprecursors capable of being polymerized or gelled. Unique identifiers,such as barcodes, may be injected into the droplets previous to,subsequent to, or concurrently with droplet generation, such as via amicrocapsule (e.g., bead), as described elsewhere herein. Microfluidicchannel networks (e.g., on a chip) can be utilized to generatepartitions as described herein. Alternative mechanisms may also beemployed in the partitioning of individual analyte carriers, includingporous membranes through which aqueous mixtures of cells are extrudedinto non-aqueous fluids.

The partitions can be flowable within fluid streams. The partitions maycomprise, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionsmay comprise a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can be droplets ofa first phase within a second phase, wherein the first and second phasesare immiscible. For example, the partitions can be droplets of aqueousfluid within a non-aqueous continuous phase (e.g., oil phase). Inanother example, the partitions can be droplets of a non-aqueous fluidwithin an aqueous phase. In some examples, the partitions may beprovided in a water-in-oil emulsion or oil-in-water emulsion. A varietyof different vessels are described in, for example, U.S. PatentApplication Publication No. 2014/0155295, which is entirely incorporatedherein by reference for all purposes. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are describedin, for example, U.S. Patent Application Publication No. 2010/0105112,which is entirely incorporated herein by reference for all purposes.

In the case of droplets in an emulsion, allocating individual particlesto discrete partitions may in one non-limiting example be accomplishedby introducing a flowing stream of particles in an aqueous fluid into aflowing stream of a non-aqueous fluid, such that droplets are generatedat the junction of the two streams. Fluid properties (e.g., fluid flowrates, fluid viscosities, etc.), particle properties (e.g., volumefraction, particle size, particle concentration, etc.), microfluidicarchitectures (e.g., channel geometry, etc.), and other parameters maybe adjusted to control the occupancy of the resulting partitions (e.g.,number of analyte carriers per partition, number of beads per partition,etc.). For example, partition occupancy can be controlled by providingthe aqueous stream at a certain concentration and/or flow rate ofparticles. To generate single analyte carrier partitions, the relativeflow rates of the immiscible fluids can be selected such that, onaverage, the partitions may contain less than one analyte carrier perpartition in order to ensure that those partitions that are occupied areprimarily singly occupied. In some cases, partitions among a pluralityof partitions may contain at most one analyte carrier (e.g., bead, DNA,cell or cellular material). In some embodiments, the various parameters(e.g., fluid properties, particle properties, microfluidicarchitectures, etc.) may be selected or adjusted such that a majority ofpartitions are occupied, for example, allowing for only a smallpercentage of unoccupied partitions. The flows and channel architecturescan be controlled as to ensure a given number of singly occupiedpartitions, less than a certain level of unoccupied partitions and/orless than a certain level of multiply occupied partitions.

FIG. 1 shows an example of a microfluidic channel structure 100 forpartitioning individual biological particles. The channel structure 100can include channel segments 102, 104, 106 and 108 communicating at achannel junction 110. In operation, a first aqueous fluid 112 thatincludes suspended biological particles (or analyte carriers, e.g.,cells) 114 may be transported along channel segment 102 into junction110, while a second fluid 116 that is immiscible with the aqueous fluid112 is delivered to the junction 110 from each of channel segments 104and 106 to create discrete droplets 118, 120 of the first aqueous fluid112 flowing into channel segment 108, and flowing away from junction110. The channel segment 108 may be fluidically coupled to an outletreservoir where the discrete droplets can be stored and/or harvested. Adiscrete droplet generated may include an individual biological particle114 (such as droplets 118). A discrete droplet generated may includemore than one individual biological particle 114 (not shown in FIG. 1).A discrete droplet may contain no biological particle 114 (such asdroplet 120). Each discrete partition may maintain separation of its owncontents (e.g., individual biological particle 114) from the contents ofother partitions.

The second fluid 116 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets118, 120. Examples of particularly useful partitioning fluids andfluorosurfactants are described, for example, in U.S. Patent ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 100 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junction.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying particles (e.g., biological particles oranalyte carriers, cell beads, and/or gel beads) that meet at a channeljunction. Fluid may be directed to flow along one or more channels orreservoirs via one or more fluid flow units. A fluid flow unit cancomprise compressors (e.g., providing positive pressure), pumps (e.g.,providing negative pressure), actuators, and the like to control flow ofthe fluid. Fluid may also or otherwise be controlled via appliedpressure differentials, centrifugal force, electrokinetic pumping,vacuum, capillary or gravity flow, or the like.

The generated droplets may comprise two subsets of droplets: (1)occupied droplets 118, containing one or more biological particles 114(or analyte carriers), and (2) unoccupied droplets 120, not containingany biological particles 114. Occupied droplets 118 may comprise singlyoccupied droplets (having one biological particle) and multiply occupieddroplets (having more than one biological particle). As describedelsewhere herein, in some cases, the majority of occupied partitions caninclude no more than one biological particle per occupied partition andsome of the generated partitions can be unoccupied (of any biologicalparticle). In some cases, though, some of the occupied partitions mayinclude more than one biological particle. In some cases, thepartitioning process may be controlled such that fewer than about 25% ofthe occupied partitions contain more than one biological particle, andin many cases, fewer than about 20% of the occupied partitions have morethan one biological particle, while in some cases, fewer than about 10%or even fewer than about 5% of the occupied partitions include more thanone biological particle per partition.

In some cases, it may be desirable to minimize the creation of excessivenumbers of empty partitions, such as to reduce costs and/or increaseefficiency. While this minimization may be achieved by providing asufficient number of biological particles (e.g., biological particles114) at the partitioning junction 110, such as to ensure that at leastone biological particle is encapsulated in a partition, the Poissoniandistribution may expectedly increase the number of partitions thatinclude multiple biological particles. As such, where singly occupiedpartitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% orless of the generated partitions can be unoccupied.

In some cases, the flow of one or more of the biological particles (oranalyte carriers) (e.g., in channel segment 102), or other fluidsdirected into the partitioning junction (e.g., in channel segments 104,106) can be controlled such that, in many cases, no more than about 50%of the generated partitions, no more than about 25% of the generatedpartitions, or no more than about 10% of the generated partitions areunoccupied. These flows can be controlled so as to present anon-Poissonian distribution of single-occupied partitions whileproviding lower levels of unoccupied partitions. The above noted rangesof unoccupied partitions can be achieved while still providing any ofthe single occupancy rates described above. For example, in many cases,the use of the systems and methods described herein can create resultingpartitions that have multiple occupancy rates of less than about 25%,less than about 20%, less than about 15%, less than about 10%, and inmany cases, less than about 5%, while having unoccupied partitions ofless than about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 10%, less than about 5%, or less.

As will be appreciated, the above-described occupancy rates are alsoapplicable to partitions that include both biological particles (oranalyte carriers) and additional reagents, including, but not limitedto, microcapsules or beads (e.g., gel beads) carrying barcoded nucleicacid molecules (e.g., oligonucleotides) (described in relation to FIG.2). The occupied partitions (e.g., at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, or 99% of the occupied partitions) caninclude both a microcapsule (e.g., bead) comprising barcoded nucleicacid molecules and a biological particle.

In another aspect, in addition to or as an alternative to droplet basedpartitioning, biological particles (or analyte carriers) may beencapsulated within a microcapsule that comprises an outer shell, layeror porous matrix in which is entrained one or more individual biologicalparticles or small groups of biological particles. The microcapsule mayinclude other reagents. Encapsulation of biological particles may beperformed by a variety of processes. Such processes may combine anaqueous fluid containing the biological particles with a polymericprecursor material that may be capable of being formed into a gel orother solid or semi-solid matrix upon application of a particularstimulus to the polymer precursor. Such stimuli can include, forexample, thermal stimuli (e.g., either heating or cooling),photo-stimuli (e.g., through photo-curing), chemical stimuli (e.g.,through crosslinking, polymerization initiation of the precursor (e.g.,through added initiators)), mechanical stimuli, or a combinationthereof.

Preparation of microcapsules comprising biological particles (or analytecarriers) may be performed by a variety of methods. For example, airknife droplet or aerosol generators may be used to dispense droplets ofprecursor fluids into gelling solutions in order to form microcapsulesthat include individual biological particles or small groups ofbiological particles. Likewise, membrane based encapsulation systems maybe used to generate microcapsules comprising encapsulated biologicalparticles as described herein. Microfluidic systems of the presentdisclosure, such as that shown in FIG. 1, may be readily used inencapsulating cells as described herein. In particular, and withreference to FIG. 1, the aqueous fluid 112 comprising (i) the biologicalparticles 114 and (ii) the polymer precursor material (not shown) isflowed into channel junction 110, where it is partitioned into droplets118, 120 through the flow of non-aqueous fluid 116. In the case ofencapsulation methods, non-aqueous fluid 116 may also include aninitiator (not shown) to cause polymerization and/or crosslinking of thepolymer precursor to form the microcapsule that includes the entrainedbiological particles. Examples of polymer precursor/initiator pairsinclude those described in U.S. Patent Application Publication No.2014/0378345, which is entirely incorporated herein by reference for allpurposes.

For example, in the case where the polymer precursor material comprisesa linear polymer material, such as a linear polyacrylamide, PEG, orother linear polymeric material, the activation agent may comprise across-linking agent, or a chemical that activates a cross-linking agentwithin the formed droplets. Likewise, for polymer precursors thatcomprise polymerizable monomers, the activation agent may comprise apolymerization initiator. For example, in certain cases, where thepolymer precursor comprises a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) may be provided within the secondfluid streams 116 in channel segments 104 and 106, which can initiatethe copolymerization of the acrylamide and BAC into a cross-linkedpolymer network, or hydrogel.

Upon contact of the second fluid stream 116 with the first fluid stream112 at junction 110, during formation of droplets, the TEMED may diffusefrom the second fluid 116 into the aqueous fluid 112 comprising thelinear polyacrylamide, which will activate the crosslinking of thepolyacrylamide within the droplets 118, 120, resulting in the formationof gel (e.g., hydrogel) microcapsules, as solid or semi-solid beads orparticles entraining the cells 114. Although described in terms ofpolyacrylamide encapsulation, other ‘activatable’ encapsulationcompositions may also be employed in the context of the methods andcompositions described herein. For example, formation of alginatedroplets followed by exposure to divalent metal ions (e.g., Ca²⁺ ions),can be used as an encapsulation process using the described processes.Likewise, agarose droplets may also be transformed into capsules throughtemperature based gelling (e.g., upon cooling, etc.).

In some cases, encapsulated biological particles (or analyte carriers)can be selectively releasable from the microcapsule, such as throughpassage of time or upon application of a particular stimulus, thatdegrades the microcapsule sufficiently to allow the biological particles(e.g., cell), or its other contents to be released from themicrocapsule, such as into a partition (e.g., droplet). For example, inthe case of the polyacrylamide polymer described above, degradation ofthe microcapsule may be accomplished through the introduction of anappropriate reducing agent, such as DTT or the like, to cleave disulfidebonds that cross-link the polymer matrix. See, for example, U.S. PatentApplication Publication No. 2014/0378345, which is entirely incorporatedherein by reference for all purposes.

The biological particle (or analyte carrier) can be subjected to otherconditions sufficient to polymerize or gel the precursors. Theconditions sufficient to polymerize or gel the precursors may compriseexposure to heating, cooling, electromagnetic radiation, and/or light.The conditions sufficient to polymerize or gel the precursors maycomprise any conditions sufficient to polymerize or gel the precursors.Following polymerization or gelling, a polymer or gel may be formedaround the biological particle. The polymer or gel may be diffusivelypermeable to chemical or biochemical reagents. The polymer or gel may bediffusively impermeable to macromolecular constituents of the biologicalparticle. In this manner, the polymer or gel may act to allow thebiological particle to be subjected to chemical or biochemicaloperations while spatially confining the macromolecular constituents toa region of the droplet defined by the polymer or gel. The polymer orgel may include one or more of disulfide cross-linked polyacrylamide,agarose, alginate, polyvinyl alcohol, polyethylene glycol(PEG)-diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG-alkyne, otheracrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, orelastin. The polymer or gel may comprise any other polymer or gel.

The polymer or gel may be functionalized to bind to targeted analytes,such as nucleic acids, proteins, carbohydrates, lipids or otheranalytes. The polymer or gel may be polymerized or gelled via a passivemechanism. The polymer or gel may be stable in alkaline conditions or atelevated temperature. The polymer or gel may have mechanical propertiessimilar to the mechanical properties of the bead. For instance, thepolymer or gel may be of a similar size to the bead. The polymer or gelmay have a mechanical strength (e.g. tensile strength) similar to thatof the bead. The polymer or gel may be of a lower density than an oil.The polymer or gel may be of a density that is roughly similar to thatof a buffer. The polymer or gel may have a tunable pore size. The poresize may be chosen to, for instance, retain denatured nucleic acids. Thepore size may be chosen to maintain diffusive permeability to exogenouschemicals such as sodium hydroxide (NaOH) and/or endogenous chemicalssuch as inhibitors. The polymer or gel may be biocompatible. The polymeror gel may maintain or enhance cell viability. The polymer or gel may bebiochemically compatible. The polymer or gel may be polymerized and/ordepolymerized thermally, chemically, enzymatically, and/or optically.

The polymer may comprise poly(acrylamide-co-acrylic acid) crosslinkedwith disulfide linkages. The preparation of the polymer may comprise atwo-step reaction. In the first activation step,poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent toconvert carboxylic acids to esters. For instance, thepoly(acrylamide-co-acrylic acid) may be exposed to4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). The polyacrylamide-co-acrylic acid may be exposed to othersalts of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. Inthe second cross-linking step, the ester formed in the first step may beexposed to a disulfide crosslinking agent. For instance, the ester maybe exposed to cystamine (2,2′-dithiobis(ethylamine)). Following the twosteps, the biological particle (or analyte carrier, such as a cell) maybe surrounded by polyacrylamide strands linked together by disulfidebridges. In this manner, the biological particle may be encased insideof or comprise a gel or matrix (e.g., polymer matrix) to form a “cellbead.” A cell bead can contain biological particles (e.g., a cell) ormacromolecular constituents (e.g., RNA, DNA, proteins, etc.) ofbiological particles. A cell bead may include a single cell or multiplecells, or a derivative of the single cell or multiple cells. A cell beadmay include a single nucleus. A cell bead may include a single,permeabilized nucleus. For example after lysing and washing the cells,inhibitory components from cell lysates can be washed away and themacromolecular constituents can be bound as cell beads. Systems andmethods disclosed herein can be applicable to both cell beads (and/ordroplets or other partitions) containing biological particles and cellbeads (and/or droplets or other partitions) containing macromolecularconstituents of biological particles. Cell beads may be or include acell, cell derivative, cellular material and/or material derived fromthe cell in, within, or encased in a matrix, such as a polymeric matrix.In some cases, a cell bead may comprise a live cell. In some instances,the live cell may be capable of being cultured when enclosed in a gel orpolymer matrix, or of being cultured when comprising a gel or polymermatrix. In some instances, the polymer or gel may be diffusivelypermeable to certain components and diffusively impermeable to othercomponents (e.g., macromolecular constituents). A cell bead may comprisenucleic acid molecules comprising one or more functional sequences(e.g., 908, 909, and/or 910).

Encapsulated biological particles (or analyte carriers) can providecertain potential advantages of being more storable and more portablethan droplet-based partitioned biological particles. Furthermore, insome cases, it may be desirable to allow biological particles toincubate for a select period of time before analysis, such as in orderto characterize changes in such biological particles over time, eitherin the presence or absence of different stimuli. In such cases,encapsulation may allow for longer incubation than partitioning inemulsion droplets, although in some cases, droplet partitionedbiological particles may also be incubated for different periods oftime, e.g., at least 10 seconds, at least 30 seconds, at least 1 minute,at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1hour, at least 2 hours, at least 5 hours, or at least 10 hours or more.The encapsulation of biological particles may constitute thepartitioning of the biological particles into which other reagents areco-partitioned. Alternatively or in addition, encapsulated biologicalparticles may be readily deposited into other partitions (e.g.,droplets) as described above.

Beads

A partition may comprise one or more unique identifiers, such asbarcodes. Barcodes may be previously, subsequently or concurrentlydelivered to the partitions that hold the compartmentalized orpartitioned analyte carrier. For example, barcodes may be injected intodroplets previous to, subsequent to, or concurrently with dropletgeneration. The delivery of the barcodes to a particular partitionallows for the later attribution of the characteristics of theindividual analyte carrier to the particular partition. Barcodes may bedelivered, for example on a nucleic acid molecule (e.g., anoligonucleotide), to a partition via any suitable mechanism. Barcodednucleic acid molecules can be delivered to a partition via amicrocapsule. A microcapsule, in some instances, can comprise a bead.Beads are described in further detail below.

In some cases, barcoded nucleic acid molecules can be initiallyassociated with the microcapsule and then released from themicrocapsule. Release of the barcoded nucleic acid molecules can bepassive (e.g., by diffusion out of the microcapsule). In addition oralternatively, release from the microcapsule can be upon application ofa stimulus which allows the barcoded nucleic acid nucleic acid moleculesto dissociate or to be released from the microcapsule. Such stimulus maydisrupt the microcapsule, an interaction that couples the barcodednucleic acid molecules to or within the microcapsule, or both. Suchstimulus can include, for example, a thermal stimulus, photo-stimulus,chemical stimulus (e.g., change in pH or use of a reducing agent(s)), amechanical stimulus, a radiation stimulus; a biological stimulus (e.g.,enzyme), or any combination thereof.

FIG. 2 shows an example of a microfluidic channel structure 200 fordelivering barcode carrying beads to droplets. The channel structure 200can include channel segments 201, 202, 204, 206 and 208 communicating ata channel junction 210. In operation, the channel segment 201 maytransport an aqueous fluid 212 that includes a plurality of beads 214(e.g., with nucleic acid molecules, oligonucleotides, molecular tags)along the channel segment 201 into junction 210. The plurality of beads214 may be sourced from a suspension of beads. For example, the channelsegment 201 may be connected to a reservoir comprising an aqueoussuspension of beads 214. The channel segment 202 may transport theaqueous fluid 212 that includes a plurality of biological particles 216(or analyte carriers) along the channel segment 202 into junction 210.The plurality of biological particles 216 may be sourced from asuspension of biological particles. For example, the channel segment 202may be connected to a reservoir comprising an aqueous suspension ofbiological particles 216. In some instances, the aqueous fluid 212 ineither the first channel segment 201 or the second channel segment 202,or in both segments, can include one or more reagents, as furtherdescribed below. A second fluid 218 that is immiscible with the aqueousfluid 212 (e.g., oil) can be delivered to the junction 210 from each ofchannel segments 204 and 206. Upon meeting of the aqueous fluid 212 fromeach of channel segments 201 and 202 and the second fluid 218 from eachof channel segments 204 and 206 at the channel junction 210, the aqueousfluid 212 can be partitioned as discrete droplets 220 in the secondfluid 218 and flow away from the junction 210 along channel segment 208.The channel segment 208 may deliver the discrete droplets to an outletreservoir fluidly coupled to the channel segment 208, where they may beharvested.

As an alternative, the channel segments 201 and 202 may meet at anotherjunction upstream of the junction 210. At such junction, beads andbiological particles may form a mixture that is directed along anotherchannel to the junction 210 to yield droplets 220. The mixture mayprovide the beads and biological particles in an alternating fashion,such that, for example, a droplet comprises a single bead and a singlebiological particle.

Beads, biological particles and droplets may flow along channels atsubstantially regular flow profiles (e.g., at regular flow rates). Suchregular flow profiles may permit a droplet to include a single bead anda single biological particle. Such regular flow profiles may permit thedroplets to have an occupancy (e.g., droplets having beads andbiological particles) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95%. Such regular flow profiles and devices that maybe used to provide such regular flow profiles are provided in, forexample, U.S. Patent Publication No. 2015/0292988, which is entirelyincorporated herein by reference.

The second fluid 218 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets220.

A discrete droplet that is generated may include an individualbiological particle 216. A discrete droplet that is generated mayinclude a barcode or other reagent carrying bead 214. A discrete dropletgenerated may include both an individual biological particle and abarcode carrying bead, such as droplets 220. In some instances, adiscrete droplet may include more than one individual biologicalparticle or no biological particle. In some instances, a discretedroplet may include more than one bead or no bead. A discrete dropletmay be unoccupied (e.g., no beads, no biological particles).

Beneficially, a discrete droplet partitioning a biological particle (oranalyte carrier) and a barcode carrying bead may effectively allow theattribution of the barcode to macromolecular constituents of thebiological particle within the partition. The contents of a partitionmay remain discrete from the contents of other partitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 200 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying beads that meet at a channel junction.Fluid may be directed flow along one or more channels or reservoirs viaone or more fluid flow units. A fluid flow unit can comprise compressors(e.g., providing positive pressure), pumps (e.g., providing negativepressure), actuators, and the like to control flow of the fluid. Fluidmay also or otherwise be controlled via applied pressure differentials,centrifugal force, electrokinetic pumping, vacuum, capillary or gravityflow, or the like.

A bead may be porous, non-porous, solid, semi-solid, semi-fluidic,fluidic, and/or a combination thereof. In some instances, a bead may bedissolvable, disruptable, and/or degradable. In some cases, a bead maynot be degradable. In some cases, the bead may be a gel bead. A gel beadmay be a hydrogel bead. A gel bead may be formed from molecularprecursors, such as a polymeric or monomeric species. A semi-solid beadmay be a liposomal bead. Solid beads may comprise metals including ironoxide, gold, and silver. In some cases, the bead may be a silica bead.In some cases, the bead can be rigid. In other cases, the bead may beflexible and/or compressible.

A bead may be of any suitable shape. Examples of bead shapes include,but are not limited to, spherical, non-spherical, oval, oblong,amorphous, circular, cylindrical, and variations thereof.

Beads may be of uniform size or heterogeneous size. In some cases, thediameter of a bead may be at least about 10 nanometers (nm), 100 nm, 500nm, 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm,70 μm, 80 μm, 90 μm, 100 m, 250 μm, 500 m, 1 mm, or greater. In somecases, a bead may have a diameter of less than about 10 nm, 100 nm, 500nm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm,90 μm, 100 μm, 250 μm, 500 μm, 1 mm, or less. In some cases, a bead mayhave a diameter in the range of about 40-75 μm, 30-75 μm, 20-75 μm,40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500μm.

In certain aspects, beads can be provided as a population or pluralityof beads having a relatively monodisperse size distribution. Where itmay be desirable to provide relatively consistent amounts of reagentswithin partitions, maintaining relatively consistent beadcharacteristics, such as size, can contribute to the overallconsistency. In particular, the beads described herein may have sizedistributions that have a coefficient of variation in theircross-sectional dimensions of less than 50%, less than 40%, less than30%, less than 20%, and in some cases less than 15%, less than 10%, lessthan 5%, or less.

A bead may comprise natural and/or synthetic materials. For example, abead can comprise a natural polymer, a synthetic polymer or both naturaland synthetic polymers. Examples of natural polymers include proteinsand sugars such as deoxyribonucleic acid, rubber, cellulose, starch(e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silks,polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan,ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum,Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate,or natural polymers thereof. Examples of synthetic polymers includeacrylics, nylons, silicones, spandex, viscose rayon, polycarboxylicacids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethyleneglycol, polyurethanes, polylactic acid, silica, polystyrene,polyacrylonitrile, polybutadiene, polycarbonate, polyethylene,polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethyleneoxide), poly(ethylene terephthalate), polyethylene, polyisobutylene,poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde,polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinylacetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidenedichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and/orcombinations (e.g., co-polymers) thereof. Beads may also be formed frommaterials other than polymers, including lipids, micelles, ceramics,glass-ceramics, material composites, metals, other inorganic materials,and others.

In some instances, the bead may contain molecular precursors (e.g.,monomers or polymers), which may form a polymer network viapolymerization of the molecular precursors. In some cases, a precursormay be an already polymerized species capable of undergoing furtherpolymerization via, for example, a chemical cross-linkage. In somecases, a precursor can comprise one or more of an acrylamide or amethacrylamide monomer, oligomer, or polymer. In some cases, the beadmay comprise prepolymers, which are oligomers capable of furtherpolymerization. For example, polyurethane beads may be prepared usingprepolymers. In some cases, the bead may contain individual polymersthat may be further polymerized together. In some cases, beads may begenerated via polymerization of different precursors, such that theycomprise mixed polymers, co-polymers, and/or block co-polymers. In somecases, the bead may comprise covalent or ionic bonds between polymericprecursors (e.g., monomers, oligomers, linear polymers), nucleic acidmolecules (e.g., oligonucleotides), primers, and other entities. In somecases, the covalent bonds can be carbon-carbon bonds, thioether bonds,or carbon-heteroatom bonds.

Cross-linking may be permanent or reversible, depending upon theparticular cross-linker used. Reversible cross-linking may allow for thepolymer to linearize or dissociate under appropriate conditions. In somecases, reversible cross-linking may also allow for reversible attachmentof a material bound to the surface of a bead. In some cases, across-linker may form disulfide linkages. In some cases, the chemicalcross-linker forming disulfide linkages may be cystamine or a modifiedcystamine.

In some cases, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and nucleic acid molecules (e.g.,oligonucleotides). Cystamine (including modified cystamines), forexample, is an organic agent comprising a disulfide bond that may beused as a crosslinker agent between individual monomeric or polymericprecursors of a bead. Polyacrylamide may be polymerized in the presenceof cystamine or a species comprising cystamine (e.g., a modifiedcystamine) to generate polyacrylamide gel beads comprising disulfidelinkages (e.g., chemically degradable beads comprisingchemically-reducible cross-linkers). The disulfide linkages may permitthe bead to be degraded (or dissolved) upon exposure of the bead to areducing agent.

In some cases, chitosan, a linear polysaccharide polymer, may becrosslinked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers may be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some cases, a bead may comprise an acrydite moiety, which in certainaspects may be used to attach one or more nucleic acid molecules (e.g.,barcode sequence, barcoded nucleic acid molecule, barcodedoligonucleotide, primer, or other oligonucleotide) to the bead. In somecases, an acrydite moiety can refer to an acrydite analogue generatedfrom the reaction of acrydite with one or more species, such as, thereaction of acrydite with other monomers and cross-linkers during apolymerization reaction. Acrydite moieties may be modified to formchemical bonds with a species to be attached, such as a nucleic acidmolecule (e.g., barcode sequence, barcoded nucleic acid molecule,barcoded oligonucleotide, primer, or other oligonucleotide). Acryditemoieties may be modified with thiol groups capable of forming adisulfide bond or may be modified with groups already comprising adisulfide bond. The thiol or disulfide (via disulfide exchange) may beused as an anchor point for a species to be attached or another part ofthe acrydite moiety may be used for attachment. In some cases,attachment can be reversible, such that when the disulfide bond isbroken (e.g., in the presence of a reducing agent), the attached speciesis released from the bead. In other cases, an acrydite moiety cancomprise a reactive hydroxyl group that may be used for attachment.

Functionalization of beads for attachment of nucleic acid molecules(e.g., oligonucleotides) may be achieved through a wide range ofdifferent approaches, including activation of chemical groups within apolymer, incorporation of active or activatable functional groups in thepolymer structure, or attachment at the pre-polymer or monomer stage inbead production.

For example, precursors (e.g., monomers, cross-linkers) that arepolymerized to form a bead may comprise acrydite moieties, such thatwhen a bead is generated, the bead also comprises acrydite moieties. Theacrydite moieties can be attached to a nucleic acid molecule (e.g.,oligonucleotide), which may include a priming sequence (e.g., a primerfor amplifying target nucleic acids, random primer, primer sequence formessenger RNA) and/or one or more barcode sequences. The one morebarcode sequences may include sequences that are the same for allnucleic acid molecules coupled to a given bead and/or sequences that aredifferent across all nucleic acid molecules coupled to the given bead.The nucleic acid molecule may be incorporated into the bead.

In some cases, the nucleic acid molecule can comprise a functionalsequence, for example, for attachment to a sequencing flow cell, suchas, for example, a P5 sequence for Illumina® sequencing. In some cases,the nucleic acid molecule or derivative thereof (e.g., oligonucleotideor polynucleotide generated from the nucleic acid molecule) can compriseanother functional sequence, such as, for example, a P7 sequence forattachment to a sequencing flow cell for Illumina sequencing. In somecases, the nucleic acid molecule can comprise a barcode sequence. Insome cases, the primer can further comprise a unique molecularidentifier (UMI). In some cases, the primer can comprise an R1 primersequence for Illumina sequencing. In some cases, the primer can comprisean R2 primer sequence for Illumina sequencing. Examples of such nucleicacid molecules (e.g., oligonucleotides, polynucleotides, etc.) and usesthereof, as may be used with compositions, devices, methods and systemsof the present disclosure, are provided in U.S. Patent Pub. Nos.2014/0378345 and 2015/0376609, each of which is entirely incorporatedherein by reference.

FIG. 8 illustrates an example of a barcode carrying bead. A nucleic acidmolecule 802, such as an oligonucleotide, can be coupled to a bead 804by a releasable linkage 806, such as, for example, a disulfide linker.The same bead 804 may be coupled (e.g., via releasable linkage) to oneor more other nucleic acid molecules 818, 820. The nucleic acid molecule802 may be or comprise a barcode. As noted elsewhere herein, thestructure of the barcode may comprise a number of sequence elements. Thenucleic acid molecule 802 may comprise a functional sequence 808 thatmay be used in subsequent processing. For example, the functionalsequence 808 may include one or more of a sequencer specific flow cellattachment sequence (e.g., a P5 sequence for Illumina® sequencingsystems) and a sequencing primer sequence (e.g., a R1 primer forIllumina® sequencing systems). The nucleic acid molecule 802 maycomprise a barcode sequence 810 for use in barcoding the sample (e.g.,DNA, RNA, protein, etc.). In some cases, the barcode sequence 810 can bebead-specific such that the barcode sequence 810 is common to allnucleic acid molecules (e.g., including nucleic acid molecule 802)coupled to the same bead 804. Alternatively or in addition, the barcodesequence 810 can be partition-specific such that the barcode sequence810 is common to all nucleic acid molecules coupled to one or more beadsthat are partitioned into the same partition. The nucleic acid molecule802 may comprise a specific priming sequence 812, such as an mRNAspecific priming sequence (e.g., poly-T sequence), a targeted primingsequence, and/or a random priming sequence. The nucleic acid molecule802 may comprise an anchoring sequence 814 to ensure that the specificpriming sequence 812 hybridizes at the sequence end (e.g., of the mRNA).For example, the anchoring sequence 814 can include a random shortsequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or longersequence, which can ensure that a poly-T segment is more likely tohybridize at the sequence end of the poly-A tail of the mRNA.

The nucleic acid molecule 802 may comprise a unique molecularidentifying sequence 816 (e.g., unique molecular identifier (UMI)). Insome cases, the unique molecular identifying sequence 816 may comprisefrom about 5 to about 8 nucleotides. Alternatively, the unique molecularidentifying sequence 816 may compress less than about 5 or more thanabout 8 nucleotides. The unique molecular identifying sequence 816 maybe a unique sequence that varies across individual nucleic acidmolecules (e.g., 802, 818, 820, etc.) coupled to a single bead (e.g.,bead 804). In some cases, the unique molecular identifying sequence 816may be a random sequence (e.g., such as a random N-mer sequence). Forexample, the UMI may provide a unique identifier of the starting mRNAmolecule that was captured, in order to allow quantitation of the numberof original expressed RNA. As will be appreciated, although FIG. 8 showsthree nucleic acid molecules 802, 818, 820 coupled to the surface of thebead 804, an individual bead may be coupled to any number of individualnucleic acid molecules, for example, from one to tens to hundreds ofthousands or even millions of individual nucleic acid molecules. Therespective barcodes for the individual nucleic acid molecules cancomprise both common sequence segments or relatively common sequencesegments (e.g., 808, 810, 812, etc.) and variable or unique sequencesegments (e.g., 816) between different individual nucleic acid moleculescoupled to the same bead.

In operation, an analyte carrier (e.g., cell, DNA, RNA, etc.) can beco-partitioned along with a barcode bearing bead 804. The barcodednucleic acid molecules 802, 818, 820 can be released from the bead 804in the partition. By way of example, in the context of analyzing sampleRNA, the poly-T segment (e.g., 812) of one of the released nucleic acidmolecules (e.g., 802) can hybridize to the poly-A tail of a mRNAmolecule. Reverse transcription may result in a cDNA transcript of themRNA, but which transcript includes each of the sequence segments 808,810, 816 of the nucleic acid molecule 802. Because the nucleic acidmolecule 802 comprises an anchoring sequence 814, it will more likelyhybridize to and prime reverse transcription at the sequence end of thepoly-A tail of the mRNA. Within any given partition, all of the cDNAtranscripts of the individual mRNA molecules may include a commonbarcode sequence segment 810. However, the transcripts made from thedifferent mRNA molecules within a given partition may vary at the uniquemolecular identifying sequence 812 segment (e.g., UMI segment).Beneficially, even following any subsequent amplification of thecontents of a given partition, the number of different UMIs can beindicative of the quantity of mRNA originating from a given partition,and thus from the analyte carrier (e.g., cell). As noted above, thetranscripts can be amplified, cleaned up and sequenced to identify thesequence of the cDNA transcript of the mRNA, as well as to sequence thebarcode segment and the UMI segment. While a poly-T primer sequence isdescribed, other targeted or random priming sequences may also be usedin priming the reverse transcription reaction. Likewise, althoughdescribed as releasing the barcoded oligonucleotides into the partition,in some cases, the nucleic acid molecules bound to the bead (e.g., gelbead) may be used to hybridize and capture the mRNA on the solid phaseof the bead, for example, in order to facilitate the separation of theRNA from other cell contents.

In some instances, a bead may comprise a capture sequence or bindingsequence configured to bind to a corresponding capture sequence orbinding sequence. In some instances, a bead may comprise a plurality ofdifferent capture sequences or binding sequences configured to bind todifferent respective corresponding capture sequences or bindingsequences. For example, a bead may comprise a first subset of one ormore capture sequences each configured to bind to a first correspondingcapture sequence, a second subset of one or more capture sequences eachconfigured to bind to a second corresponding capture sequence, a thirdsubset of one or more capture sequences each configured to bind to athird corresponding capture sequence, and etc. A bead may comprise anynumber of different capture sequences. In some instances, a bead maycomprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different capturesequences or binding sequences configured to bind to differentrespective capture sequences or binding sequences, respectively.Alternatively or in addition, a bead may comprise at most about 10, 9,8, 7, 6, 5, 4, 3, or 2 different capture sequences or binding sequencesconfigured to bind to different respective capture sequences or bindingsequences. In some instances, the different capture sequences or bindingsequences may be configured to facilitate analysis of a same type ofanalyte. In some instances, the different capture sequences or bindingsequences may be configured to facilitate analysis of different types ofanalytes (with the same bead). The capture sequence may be designed toattach to a corresponding capture sequence. Beneficially, suchcorresponding capture sequence may be introduced to, or otherwiseinduced in, an analyte carrier (e.g., cell, cell bead, etc.) forperforming different assays in various formats (e.g., barcodedantibodies comprising the corresponding capture sequence, barcoded MHCdextramers comprising the corresponding capture sequence, barcoded guideRNA molecules comprising the corresponding capture sequence, etc.), suchthat the corresponding capture sequence may later interact with thecapture sequence associated with the bead.

FIG. 22 illustrates another example of a barcode carrying bead. Anucleic acid molecule 2205, such as an oligonucleotide, can be coupledto a bead 2204 by a releasable linkage 2206, such as, for example, adisulfide linker. The nucleic acid molecule 2205 may comprise a firstcapture sequence 2260. The same bead 2204 may be coupled (e.g., viareleasable linkage) to one or more other nucleic acid molecules 2203,2207 comprising other capture sequences. The nucleic acid molecule 2205may be or comprise a barcode. As noted elsewhere herein, the structureof the barcode may comprise a number of sequence elements, such as afunctional sequence 2208 (e.g., flow cell attachment sequence,sequencing primer sequence, etc.), a barcode sequence 2210 (e.g.,bead-specific sequence common to bead, partition-specific sequencecommon to partition, etc.), and a unique molecular identifier 2212(e.g., unique sequence within different molecules attached to the bead).The capture sequence 2260 may be configured to attach to a correspondingcapture sequence 2265. In some instances, the corresponding capturesequence 2265 may be coupled to another molecule that may be an analyteor an intermediary carrier. For example, as illustrated in FIG. 22, thecorresponding capture sequence 2265 is coupled to a guide RNA molecule2262 comprising a target sequence 2264, wherein the target sequence 2264is configured to attach to the analyte. Another oligonucleotide molecule2207 attached to the bead 2204 comprises a second capture sequence 2280which is configured to attach to a second corresponding capture sequence2285. As illustrated in FIG. 22, the second corresponding capturesequence 2285 is coupled to an antibody 2282. In some cases, theantibody 2282 may have binding specificity to an analyte (e.g., surfaceprotein). Alternatively, the antibody 2282 may not have bindingspecificity. Another oligonucleotide molecule 2203 attached to the bead2204 comprises a third capture sequence 2270 which is configured toattach to a second corresponding capture sequence 2275. As illustratedin FIG. 22, the third corresponding capture sequence 2275 is coupled toa molecule 2272. The molecule 2272 may or may not be configured totarget an analyte. The other oligonucleotide molecules 2203, 2207 maycomprise the other sequences (e.g., functional sequence, barcodesequence, UMI, etc.) described with respect to oligonucleotide molecule2205. While a single oligonucleotide molecule comprising each capturesequence is illustrated in FIG. 22, it will be appreciated that, foreach capture sequence, the bead may comprise a set of one or moreoligonucleotide molecules each comprising the capture sequence. Forexample, the bead may comprise any number of sets of one or moredifferent capture sequences. Alternatively or in addition, the bead 2204may comprise other capture sequences. Alternatively or in addition, thebead 2204 may comprise fewer types of capture sequences (e.g., twocapture sequences). Alternatively or in addition, the bead 2204 maycomprise oligonucleotide molecule(s) comprising a priming sequence, suchas a specific priming sequence such as an mRNA specific priming sequence(e.g., poly-T sequence), a targeted priming sequence, and/or a randompriming sequence, for example, to facilitate an assay for geneexpression.

In operation, the barcoded oligonucleotides may be released (e.g., in apartition), as described elsewhere herein. Alternatively, the nucleicacid molecules bound to the bead (e.g., gel bead) may be used tohybridize and capture analytes (e.g., one or more types of analytes) onthe solid phase of the bead.

In some cases, precursors comprising a functional group that is reactiveor capable of being activated such that it becomes reactive can bepolymerized with other precursors to generate gel beads comprising theactivated or activatable functional group. The functional group may thenbe used to attach additional species (e.g., disulfide linkers, primers,other oligonucleotides, etc.) to the gel beads. For example, someprecursors comprising a carboxylic acid (COOH) group can co-polymerizewith other precursors to form a gel bead that also comprises a COOHfunctional group. In some cases, acrylic acid (a species comprising freeCOOH groups), acrylamide, and bis(acryloyl)cystamine can beco-polymerized together to generate a gel bead comprising free COOHgroups. The COOH groups of the gel bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

Beads comprising disulfide linkages in their polymeric network may befunctionalized with additional species via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages may be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies comprising another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some cases, free thiols of the beads mayreact with any other suitable group. For example, free thiols of thebeads may react with species comprising an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species comprising the acrydite islinked to the bead. In some cases, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such asN-ethylmalieamide or iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlmay be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In somecases, a low concentration (e.g., molecules of reducing agent:gel beadratios of less than or equal to about 1:100,000,000,000, less than orequal to about 1:10,000,000,000, less than or equal to about1:1,000,000,000, less than or equal to about 1:100,000,000, less than orequal to about 1:10,000,000, less than or equal to about 1:1,000,000,less than or equal to about 1:100,000, less than or equal to about1:10,000) of reducing agent may be used for reduction. Controlling thenumber of disulfide linkages that are reduced to free thiols may beuseful in ensuring bead structural integrity during functionalization.In some cases, optically-active agents, such as fluorescent dyes may becoupled to beads via free thiol groups of the beads and used to quantifythe number of free thiols present in a bead and/or track a bead.

In some cases, addition of moieties to a gel bead after gel beadformation may be advantageous. For example, addition of anoligonucleotide (e.g., barcoded oligonucleotide) after gel beadformation may avoid loss of the species during chain transfertermination that can occur during polymerization. Moreover, smallerprecursors (e.g., monomers or cross linkers that do not comprise sidechain groups and linked moieties) may be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome cases, functionalization after gel bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some cases, the generated gel may possess an uppercritical solution temperature (UCST) that can permit temperature drivenswelling and collapse of a bead. Such functionality may aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide.Post-production functionalization may also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized. Species loading may also beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

A bead injected or otherwise introduced into a partition may comprisereleasably, cleavably, or reversibly attached barcodes. A bead injectedor otherwise introduced into a partition may comprise activatablebarcodes. A bead injected or otherwise introduced into a partition maybe degradable, disruptable, or dissolvable beads.

Barcodes can be releasably, cleavably or reversibly attached to thebeads such that barcodes can be released or be releasable throughcleavage of a linkage between the barcode molecule and the bead, orreleased through degradation of the underlying bead itself, allowing thebarcodes to be accessed or be accessible by other reagents, or both. Innon-limiting examples, cleavage may be achieved through reduction ofdi-sulfide bonds, use of restriction enzymes, photo-activated cleavage,or cleavage via other types of stimuli (e.g., chemical, thermal, pH,enzymatic, etc.) and/or reactions, such as described elsewhere herein.Releasable barcodes may sometimes be referred to as being activatable,in that they are available for reaction once released. Thus, forexample, an activatable barcode may be activated by releasing thebarcode from a bead (or other suitable type of partition describedherein). Other activatable configurations are also envisioned in thecontext of the described methods and systems.

In addition to, or as an alternative to the cleavable linkages betweenthe beads and the associated molecules, such as barcode containingnucleic acid molecules (e.g., barcoded oligonucleotides), the beads maybe degradable, disruptable, or dissolvable spontaneously or uponexposure to one or more stimuli (e.g., temperature changes, pH changes,exposure to particular chemical species or phase, exposure to light,reducing agent, etc.). In some cases, a bead may be dissolvable, suchthat material components of the beads are solubilized when exposed to aparticular chemical species or an environmental change, such as a changetemperature or a change in pH. In some cases, a gel bead can be degradedor dissolved at elevated temperature and/or in basic conditions. In somecases, a bead may be thermally degradable such that when the bead isexposed to an appropriate change in temperature (e.g., heat), the beaddegrades. Degradation or dissolution of a bead bound to a species (e.g.,a nucleic acid molecule, e.g., barcoded oligonucleotide) may result inrelease of the species from the bead.

As will be appreciated from the above disclosure, the degradation of abead may refer to the disassociation of a bound or entrained speciesfrom a bead, both with and without structurally degrading the physicalbead itself. For example, the degradation of the bead may involvecleavage of a cleavable linkage via one or more species and/or methodsdescribed elsewhere herein. In another example, entrained species may bereleased from beads through osmotic pressure differences due to, forexample, changing chemical environments. By way of example, alterationof bead pore sizes due to osmotic pressure differences can generallyoccur without structural degradation of the bead itself. In some cases,an increase in pore size due to osmotic swelling of a bead can permitthe release of entrained species within the bead. In other cases,osmotic shrinking of a bead may cause a bead to better retain anentrained species due to pore size contraction.

A degradable bead may be introduced into a partition, such as a dropletof an emulsion or a well, such that the bead degrades within thepartition and any associated species (e.g., oligonucleotides) arereleased within the droplet when the appropriate stimulus is applied.The free species (e.g., oligonucleotides, nucleic acid molecules) mayinteract with other reagents contained in the partition. For example, apolyacrylamide bead comprising cystamine and linked, via a disulfidebond, to a barcode sequence, may be combined with a reducing agentwithin a droplet of a water-in-oil emulsion. Within the droplet, thereducing agent can break the various disulfide bonds, resulting in beaddegradation and release of the barcode sequence into the aqueous, innerenvironment of the droplet. In another example, heating of a dropletcomprising a bead-bound barcode sequence in basic solution may alsoresult in bead degradation and release of the attached barcode sequenceinto the aqueous, inner environment of the droplet.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingnucleic acid molecule (e.g., oligonucleotide) bearing beads.

In some cases, beads can be non-covalently loaded with one or morereagents. The beads can be non-covalently loaded by, for instance,subjecting the beads to conditions sufficient to swell the beads,allowing sufficient time for the reagents to diffuse into the interiorsof the beads, and subjecting the beads to conditions sufficient tode-swell the beads. The swelling of the beads may be accomplished, forinstance, by placing the beads in a thermodynamically favorable solvent,subjecting the beads to a higher or lower temperature, subjecting thebeads to a higher or lower ion concentration, and/or subjecting thebeads to an electric field. The swelling of the beads may beaccomplished by various swelling methods. The de-swelling of the beadsmay be accomplished, for instance, by transferring the beads in athermodynamically unfavorable solvent, subjecting the beads to lower orhigh temperatures, subjecting the beads to a lower or higher ionconcentration, and/or removing an electric field. The de-swelling of thebeads may be accomplished by various de-swelling methods. Transferringthe beads may cause pores in the bead to shrink. The shrinking may thenhinder reagents within the beads from diffusing out of the interiors ofthe beads. The hindrance may be due to steric interactions between thereagents and the interiors of the beads. The transfer may beaccomplished microfluidically. For instance, the transfer may beachieved by moving the beads from one co-flowing solvent stream to adifferent co-flowing solvent stream. The swellability and/or pore sizeof the beads may be adjusted by changing the polymer composition of thebead.

In some cases, an acrydite moiety linked to a precursor, another specieslinked to a precursor, or a precursor itself can comprise a labile bond,such as chemically, thermally, or photo-sensitive bond e.g., disulfidebond, UV sensitive bond, or the like. Once acrydite moieties or othermoieties comprising a labile bond are incorporated into a bead, the beadmay also comprise the labile bond. The labile bond may be, for example,useful in reversibly linking (e.g., covalently linking) species (e.g.,barcodes, primers, etc.) to a bead. In some cases, a thermally labilebond may include a nucleic acid hybridization based attachment, e.g.,where an oligonucleotide is hybridized to a complementary sequence thatis attached to the bead, such that thermal melting of the hybridreleases the oligonucleotide, e.g., a barcode containing sequence, fromthe bead or microcapsule.

The addition of multiple types of labile bonds to a gel bead may resultin the generation of a bead capable of responding to varied stimuli.Each type of labile bond may be sensitive to an associated stimulus(e.g., chemical stimulus, light, temperature, enzymatic, etc.) such thatrelease of species attached to a bead via each labile bond may becontrolled by the application of the appropriate stimulus. Suchfunctionality may be useful in controlled release of species from a gelbead. In some cases, another species comprising a labile bond may belinked to a gel bead after gel bead formation via, for example, anactivated functional group of the gel bead as described above. As willbe appreciated, barcodes that are releasably, cleavably or reversiblyattached to the beads described herein include barcodes that arereleased or releasable through cleavage of a linkage between the barcodemolecule and the bead, or that are released through degradation of theunderlying bead itself, allowing the barcodes to be accessed oraccessible by other reagents, or both.

The barcodes that are releasable as described herein may sometimes bereferred to as being activatable, in that they are available forreaction once released. Thus, for example, an activatable barcode may beactivated by releasing the barcode from a bead (or other suitable typeof partition described herein). Other activatable configurations arealso envisioned in the context of the described methods and systems.

In addition to thermally cleavable bonds, disulfide bonds and UVsensitive bonds, other non-limiting examples of labile bonds that may becoupled to a precursor or bead include an ester linkage (e.g., cleavablewith an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g.,cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavablevia heat), a sulfone linkage (e.g., cleavable via a base), a silyl etherlinkage (e.g., cleavable via an acid), a glycosidic linkage (e.g.,cleavable via an amylase), a peptide linkage (e.g., cleavable via aprotease), or a phosphodiester linkage (e.g., cleavable via a nuclease(e.g., DNase)). A bond may be cleavable via other nucleic acid moleculetargeting enzymes, such as restriction enzymes (e.g., restrictionendonucleases), as described further below.

Species may be encapsulated in beads during bead generation (e.g.,during polymerization of precursors). Such species may or may notparticipate in polymerization. Such species may be entered intopolymerization reaction mixtures such that generated beads comprise thespecies upon bead formation. In some cases, such species may be added tothe gel beads after formation. Such species may include, for example,nucleic acid molecules (e.g., oligonucleotides), reagents for a nucleicacid amplification reaction (e.g., primers, polymerases, dNTPs,co-factors (e.g., ionic co-factors), buffers) including those describedherein, reagents for enzymatic reactions (e.g., enzymes, co-factors,substrates, buffers), reagents for nucleic acid modification reactionssuch as polymerization, ligation, or digestion, and/or reagents fortemplate preparation (e.g., tagmentation) for one or more sequencingplatforms (e.g., Nextera® for Illumina®). Such species may include oneor more enzymes described herein, including without limitation,polymerase, reverse transcriptase, restriction enzymes (e.g.,endonuclease), transposase, ligase, proteinase K, DNAse, etc. Suchspecies may include one or more reagents described elsewhere herein(e.g., lysis agents, inhibitors, inactivating agents, chelating agents,stimulus). Trapping of such species may be controlled by the polymernetwork density generated during polymerization of precursors, controlof ionic charge within the gel bead (e.g., via ionic species linked topolymerized species), or by the release of other species. Encapsulatedspecies may be released from a bead upon bead degradation and/or byapplication of a stimulus capable of releasing the species from thebead. Alternatively or in addition, species may be partitioned in apartition (e.g., droplet) during or subsequent to partition formation.Such species may include, without limitation, the abovementioned speciesthat may also be encapsulated in a bead.

A degradable bead may comprise one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimuli,the bond is broken and the bead degrades. The labile bond may be achemical bond (e.g., covalent bond, ionic bond) or may be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some cases, a crosslinker used to generate abead may comprise a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead comprising cystaminecrosslinkers to a reducing agent, the disulfide bonds of the cystaminecan be broken and the bead degraded.

A degradable bead may be useful in more quickly releasing an attachedspecies (e.g., a nucleic acid molecule, a barcode sequence, a primer,etc) from the bead when the appropriate stimulus is applied to the beadas compared to a bead that does not degrade. For example, for a speciesbound to an inner surface of a porous bead or in the case of anencapsulated species, the species may have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some cases, a species may also be attached to a degradable bead via adegradable linker (e.g., disulfide linker). The degradable linker mayrespond to the same stimuli as the degradable bead or the two degradablespecies may respond to different stimuli. For example, a barcodesequence may be attached, via a disulfide bond, to a polyacrylamide beadcomprising cystamine. Upon exposure of the barcoded-bead to a reducingagent, the bead degrades and the barcode sequence is released uponbreakage of both the disulfide linkage between the barcode sequence andthe bead and the disulfide linkages of the cystamine in the bead.

As will be appreciated from the above disclosure, while referred to asdegradation of a bead, in many instances as noted above, thatdegradation may refer to the disassociation of a bound or entrainedspecies from a bead, both with and without structurally degrading thephysical bead itself. For example, entrained species may be releasedfrom beads through osmotic pressure differences due to, for example,changing chemical environments. By way of example, alteration of beadpore sizes due to osmotic pressure differences can generally occurwithout structural degradation of the bead itself. In some cases, anincrease in pore size due to osmotic swelling of a bead can permit therelease of entrained species within the bead. In other cases, osmoticshrinking of a bead may cause a bead to better retain an entrainedspecies due to pore size contraction.

Where degradable beads are provided, it may be beneficial to avoidexposing such beads to the stimulus or stimuli that cause suchdegradation prior to a given time, in order to, for example, avoidpremature bead degradation and issues that arise from such degradation,including for example poor flow characteristics and aggregation. By wayof example, where beads comprise reducible cross-linking groups, such asdisulfide groups, it will be desirable to avoid contacting such beadswith reducing agents, e.g., DTT or other disulfide cleaving reagents. Insuch cases, treatment to the beads described herein will, in some casesbe provided free of reducing agents, such as DTT. Because reducingagents are often provided in commercial enzyme preparations, it may bedesirable to provide reducing agent free (or DTT free) enzymepreparations in treating the beads described herein. Examples of suchenzymes include, e.g., polymerase enzyme preparations, reversetranscriptase enzyme preparations, ligase enzyme preparations, as wellas many other enzyme preparations that may be used to treat the beadsdescribed herein. The terms “reducing agent free” or “DTT free”preparations can refer to a preparation having less than about 1/10th,less than about 1/50th, or even less than about 1/100th of the lowerranges for such materials used in degrading the beads. For example, forDTT, the reducing agent free preparation can have less than about 0.01millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even lessthan about 0.0001 mM DTT. In many cases, the amount of DTT can beundetectable.

Numerous chemical triggers may be used to trigger the degradation ofbeads. Examples of these chemical changes may include, but are notlimited to pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead may be formed from materials that comprisedegradable chemical crosslinkers, such as BAC or cystamine. Degradationof such degradable crosslinkers may be accomplished through a number ofmechanisms. In some examples, a bead may be contacted with a chemicaldegrading agent that may induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent may be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent may degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead. In other cases, a change in pH of a solution, such as an increasein pH, may trigger degradation of a bead. In other cases, exposure to anaqueous solution, such as water, may trigger hydrolytic degradation, andthus degradation of the bead. In some cases, any combination of stimulimay trigger degradation of a bead. For example, a change in pH mayenable a chemical agent (e.g., DTT) to become an effective reducingagent.

Beads may also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat may cause melting of a bead such that a portion of thebead degrades. In other cases, heat may increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat mayalso act upon heat-sensitive polymers used as materials to constructbeads.

Any suitable agent may degrade beads. In some embodiments, changes intemperature or pH may be used to degrade thermo-sensitive orpH-sensitive bonds within beads. In some embodiments, chemical degradingagents may be used to degrade chemical bonds within beads by oxidation,reduction or other chemical changes. For example, a chemical degradingagent may be a reducing agent, such as DTT, wherein DTT may degrade thedisulfide bonds formed between a crosslinker and gel precursors, thusdegrading the bead. In some embodiments, a reducing agent may be addedto degrade the bead, which may or may not cause the bead to release itscontents. Examples of reducing agents may include dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof. The reducing agent may be present at a concentration of about0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM. The reducing agent may be present ata concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, orgreater than 10 mM. The reducing agent may be present at concentrationof at most about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingoligonucleotide bearing beads.

Although FIG. 1 and FIG. 2 have been described in terms of providingsubstantially singly occupied partitions, above, in certain cases, itmay be desirable to provide multiply occupied partitions, e.g.,containing two, three, four or more cells and/or microcapsules (e.g.,beads) comprising barcoded nucleic acid molecules (e.g.,oligonucleotides) within a single partition. Accordingly, as notedabove, the flow characteristics of the biological particle (or analytecarrier) and/or bead containing fluids and partitioning fluids may becontrolled to provide for such multiply occupied partitions. Inparticular, the flow parameters may be controlled to provide a givenoccupancy rate at greater than about 50% of the partitions, greater thanabout 75%, and in some cases greater than about 80%, 90%, 95%, orhigher.

In some cases, additional microcapsules can be used to deliveradditional reagents to a partition. In such cases, it may beadvantageous to introduce different beads into a common channel ordroplet generation junction, from different bead sources (e.g.,containing different associated reagents) through different channelinlets into such common channel or droplet generation junction (e.g.,junction 210). In such cases, the flow and frequency of the differentbeads into the channel or junction may be controlled to provide for acertain ratio of microcapsules from each source, while ensuring a givenpairing or combination of such beads into a partition with a givennumber of biological particles (e.g., one biological particle and onebead per partition).

The partitions described herein may comprise small volumes, for example,less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL),800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL,20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

For example, in the case of droplet based partitions, the droplets mayhave overall volumes that are less than about 1000 pL, 900 pL, 800 pL,700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10pL, 1 pL, or less. Where co-partitioned with microcapsules, it will beappreciated that the sample fluid volume, e.g., including co-partitionedanalyte carriers and/or beads, within the partitions may be less thanabout 90% of the above described volumes, less than about 80%, less thanabout 70%, less than about 60%, less than about 50%, less than about40%, less than about 30%, less than about 20%, or less than about 10% ofthe above described volumes.

As is described elsewhere herein, partitioning species may generate apopulation or plurality of partitions. In such cases, any suitablenumber of partitions can be generated or otherwise provided. Forexample, at least about 1,000 partitions, at least about 5,000partitions, at least about 10,000 partitions, at least about 50,000partitions, at least about 100,000 partitions, at least about 500,000partitions, at least about 1,000,000 partitions, at least about5,000,000 partitions at least about 10,000,000 partitions, at leastabout 50,000,000 partitions, at least about 100,000,000 partitions, atleast about 500,000,000 partitions, at least about 1,000,000,000partitions, or more partitions can be generated or otherwise provided.Moreover, the plurality of partitions may comprise both unoccupiedpartitions (e.g., empty partitions) and occupied partitions.

Reagents

In accordance with certain aspects, analyte carriers may be partitionedalong with lysis reagents in order to release the contents of theanalyte carriers within the partition. In such cases, the lysis agentscan be contacted with the analyte carrier suspension concurrently with,or immediately prior to, the introduction of the analyte carriers intothe partitioning junction/droplet generation zone (e.g., junction 210),such as through an additional channel or channels upstream of thechannel junction. In accordance with other aspects, additionally oralternatively, analyte carriers may be partitioned along with otherreagents, as will be described further below.

FIG. 3 shows an example of a microfluidic channel structure 300 forco-partitioning biological particles (or analyte carriers) and reagents.The channel structure 300 can include channel segments 301, 302, 304,306 and 308. Channel segments 301 and 302 communicate at a first channeljunction 309. Channel segments 302, 304, 306, and 308 communicate at asecond channel junction 310.

In an example operation, the channel segment 301 may transport anaqueous fluid 312 that includes a plurality of biological particles 314(or analyte carriers) along the channel segment 301 into the secondjunction 310. As an alternative or in addition to, channel segment 301may transport beads (e.g., gel beads). The beads may comprise barcodemolecules.

For example, the channel segment 301 may be connected to a reservoircomprising an aqueous suspension of biological particles 314. Upstreamof, and immediately prior to reaching, the second junction 310, thechannel segment 301 may meet the channel segment 302 at the firstjunction 309. The channel segment 302 may transport a plurality ofreagents 315 (e.g., lysis agents) suspended in the aqueous fluid 312along the channel segment 302 into the first junction 309. For example,the channel segment 302 may be connected to a reservoir comprising thereagents 315. After the first junction 309, the aqueous fluid 312 in thechannel segment 301 can carry both the biological particles 314 and thereagents 315 towards the second junction 310. In some instances, theaqueous fluid 312 in the channel segment 301 can include one or morereagents, which can be the same or different reagents as the reagents315. A second fluid 316 that is immiscible with the aqueous fluid 312(e.g., oil) can be delivered to the second junction 310 from each ofchannel segments 304 and 306. Upon meeting of the aqueous fluid 312 fromthe channel segment 301 and the second fluid 316 from each of channelsegments 304 and 306 at the second channel junction 310, the aqueousfluid 312 can be partitioned as discrete droplets 318 in the secondfluid 316 and flow away from the second junction 310 along channelsegment 308. The channel segment 308 may deliver the discrete droplets318 to an outlet reservoir fluidly coupled to the channel segment 308,where they may be harvested.

The second fluid 316 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets318.

A discrete droplet generated may include an individual biologicalparticle 314 and/or one or more reagents 315. In some instances, adiscrete droplet generated may include a barcode carrying bead (notshown), such as via other microfluidics structures described elsewhereherein. In some instances, a discrete droplet may be unoccupied (e.g.,no reagents, no biological particles).

Beneficially, when lysis reagents and biological particles (or analytecarriers) are co-partitioned, the lysis reagents can facilitate therelease of the contents of the biological particles within thepartition. The contents released in a partition may remain discrete fromthe contents of other partitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 300 may have other geometries. For example, amicrofluidic channel structure can have more than two channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, 5channel segments or more each carrying the same or different types ofbeads, reagents, and/or analyte carriers that meet at a channeljunction. Fluid flow in each channel segment may be controlled tocontrol the partitioning of the different elements into droplets. Fluidmay be directed flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

Examples of lysis agents include bioactive reagents, such as lysisenzymes that are used for lysis of different cell types, e.g., grampositive or negative bacteria, plants, yeast, mammalian, etc., such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other lysis enzymes available from, e.g.,Sigma-Aldrich, Inc. (St Louis, Mo.), as well as other commerciallyavailable lysis enzymes. Other lysis agents may additionally oralternatively be co-partitioned with the analyte carriers to cause therelease of the analyte carriers's contents into the partitions. Forexample, in some cases, surfactant-based lysis solutions may be used tolyse cells, although these may be less desirable for emulsion basedsystems where the surfactants can interfere with stable emulsions. Insome cases, lysis solutions may include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some cases, lysis solutionsmay include ionic surfactants such as, for example, sarcosyl and sodiumdodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanicalcellular disruption may also be used in certain cases, e.g.,non-emulsion based partitioning such as encapsulation of analytecarriers that may be in addition to or in place of droplet partitioning,where any pore size of the encapsulate is sufficiently small to retainnucleic acid fragments of a given size, following cellular disruption.

Alternatively or in addition to the lysis agents co-partitioned with theanalyte carriers described above, other reagents can also beco-partitioned with the analyte carriers, including, for example, DNaseand RNase inactivating agents or inhibitors, such as proteinase K,chelating agents, such as EDTA, and other reagents employed in removingor otherwise reducing negative activity or impact of different celllysate components on subsequent processing of nucleic acids. Inaddition, in the case of encapsulated analyte carriers, the analytecarriers may be exposed to an appropriate stimulus to release theanalyte carriers or their contents from a co-partitioned microcapsule.For example, in some cases, a chemical stimulus may be co-partitionedalong with an encapsulated analyte carrier to allow for the degradationof the microcapsule and release of the cell or its contents into thelarger partition. In some cases, this stimulus may be the same as thestimulus described elsewhere herein for release of nucleic acidmolecules (e.g., oligonucleotides) from their respective microcapsule(e.g., bead). In alternative aspects, this may be a different andnon-overlapping stimulus, in order to allow an encapsulated analytecarrier to be released into a partition at a different time from therelease of nucleic acid molecules into the same partition.

Additional reagents may also be co-partitioned with the analytecarriers, such as endonucleases to fragment an analyte carrier's DNA,DNA polymerase enzymes and dNTPs used to amplify the analyte carrier'snucleic acid fragments and to attach the barcode molecular tags to theamplified fragments. Other enzymes may be co-partitioned, includingwithout limitation, polymerase, transposase, ligase, proteinase K,DNAse, etc. Additional reagents may also include reverse transcriptaseenzymes, including enzymes with terminal transferase activity, primersand oligonucleotides, and switch oligonucleotides (also referred toherein as “switch oligos” or “template switching oligonucleotides”)which can be used for template switching. In some cases, templateswitching can be used to increase the length of a cDNA. In some cases,template switching can be used to append a predefined nucleic acidsequence to the cDNA. In an example of template switching, cDNA can begenerated from reverse transcription of a template, e.g., cellular mRNA,where a reverse transcriptase with terminal transferase activity can addadditional nucleotides, e.g., polyC, to the cDNA in a templateindependent manner. Switch oligos can include sequences complementary tothe additional nucleotides, e.g., polyG. The additional nucleotides(e.g., polyC) on the cDNA can hybridize to the additional nucleotides(e.g., polyG) on the switch oligo, whereby the switch oligo can be usedby the reverse transcriptase as template to further extend the cDNA.Template switching oligonucleotides may comprise a hybridization regionand a template region. The hybridization region can comprise anysequence capable of hybridizing to the target. In some cases, aspreviously described, the hybridization region comprises a series of Gbases to complement the overhanging C bases at the 3′ end of a cDNAmolecule. The series of G bases may comprise 1 G base, 2 G bases, 3 Gbases, 4 G bases, 5 G bases or more than 5 G bases. The templatesequence can comprise any sequence to be incorporated into the cDNA. Insome cases, the template region comprises at least 1 (e.g., at least 2,3, 4, 5 or more) tag sequences and/or functional sequences. Switcholigos may comprise deoxyribonucleic acids; ribonucleic acids; modifiednucleic acids including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA),inverted dT, 5-Methyl dC, 2′-deoxyInosine, Super T(5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine),locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A,UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ Fluoro bases (e.g., Fluoro C,Fluoro U, Fluoro A, and Fluoro G), or any combination.

In some cases, the length of a switch oligo may be at least about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides or longer.

In some cases, the length of a switch oligo may be at most about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides.

Once the contents of the cells are released into their respectivepartitions, the macromolecular components (e.g., macromolecularconstituents of analyte carriers, such as RNA, DNA, or proteins)contained therein may be further processed within the partitions. Inaccordance with the methods and systems described herein, themacromolecular component contents of individual analyte carriers can beprovided with unique identifiers such that, upon characterization ofthose macromolecular components they may be attributed as having beenderived from the same analyte carrier or particles. The ability toattribute characteristics to individual analyte carriers or groups ofanalyte carriers is provided by the assignment of unique identifiersspecifically to an individual analyte carrier or groups of analytecarriers. Unique identifiers, e.g., in the form of nucleic acid barcodescan be assigned or associated with individual analyte carriers orpopulations of analyte carriers, in order to tag or label the analytecarrier's macromolecular components (and as a result, itscharacteristics) with the unique identifiers. These unique identifierscan then be used to attribute the analyte carrier's components andcharacteristics to an individual analyte carrier or group of analytecarriers.

In some aspects, this is performed by co-partitioning the individualanalyte carrier or groups of analyte carriers with the uniqueidentifiers, such as described above (with reference to FIG. 2). In someaspects, the unique identifiers are provided in the form of nucleic acidmolecules (e.g., oligonucleotides) that comprise nucleic acid barcodesequences that may be attached to or otherwise associated with thenucleic acid contents of individual analyte carrier, or to othercomponents of the analyte carrier, and particularly to fragments ofthose nucleic acids. The nucleic acid molecules are partitioned suchthat as between nucleic acid molecules in a given partition, the nucleicacid barcode sequences contained therein are the same, but as betweendifferent partitions, the nucleic acid molecule can, and do havediffering barcode sequences, or at least represent a large number ofdifferent barcode sequences across all of the partitions in a givenanalysis. In some aspects, only one nucleic acid barcode sequence can beassociated with a given partition, although in some cases, two or moredifferent barcode sequences may be present.

The nucleic acid barcode sequences can include from about 6 to about 20or more nucleotides within the sequence of the nucleic acid molecules(e.g., oligonucleotides). The nucleic acid barcode sequences can includefrom about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or morenucleotides. In some cases, the length of a barcode sequence may beabout 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotidesor longer. In some cases, the length of a barcode sequence may be atleast about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20nucleotides or longer. In some cases, the length of a barcode sequencemay be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 nucleotides or shorter. These nucleotides may be completelycontiguous, i.e., in a single stretch of adjacent nucleotides, or theymay be separated into two or more separate subsequences that areseparated by 1 or more nucleotides. In some cases, separated barcodesubsequences can be from about 4 to about 16 nucleotides in length. Insome cases, the barcode subsequence may be about 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcodesubsequence may be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 nucleotides or longer. In some cases, the barcode subsequence maybe at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or shorter.

The co-partitioned nucleic acid molecules can also comprise otherfunctional sequences useful in the processing of the nucleic acids fromthe co-partitioned analyte carriers. These sequences include, e.g.,targeted or random/universal amplification primer sequences foramplifying the genomic DNA from the individual analyte carriers withinthe partitions while attaching the associated barcode sequences,sequencing primers or primer recognition sites, hybridization or probingsequences, e.g., for identification of presence of the sequences or forpulling down barcoded nucleic acids, or any of a number of otherpotential functional sequences. Other mechanisms of co-partitioningoligonucleotides may also be employed, including, e.g., coalescence oftwo or more droplets, where one droplet contains oligonucleotides, ormicrodispensing of oligonucleotides into partitions, e.g., dropletswithin microfluidic systems.

In an example, microcapsules, such as beads, are provided that eachinclude large numbers of the above described barcoded nucleic acidmolecules (e.g., barcoded oligonucleotides) releasably attached to thebeads, where all of the nucleic acid molecules attached to a particularbead will include the same nucleic acid barcode sequence, but where alarge number of diverse barcode sequences are represented across thepopulation of beads used. In some embodiments, hydrogel beads, e.g.,comprising polyacrylamide polymer matrices, are used as a solid supportand delivery vehicle for the nucleic acid molecules into the partitions,as they are capable of carrying large numbers of nucleic acid molecules,and may be configured to release those nucleic acid molecules uponexposure to a particular stimulus, as described elsewhere herein. Insome cases, the population of beads provides a diverse barcode sequencelibrary that includes at least about 1,000 different barcode sequences,at least about 5,000 different barcode sequences, at least about 10,000different barcode sequences, at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences, or more. Additionally, each bead can be provided withlarge numbers of nucleic acid (e.g., oligonucleotide) moleculesattached. In particular, the number of molecules of nucleic acidmolecules including the barcode sequence on an individual bead can be atleast about 1,000 nucleic acid molecules, at least about 5,000 nucleicacid molecules, at least about 10,000 nucleic acid molecules, at leastabout 50,000 nucleic acid molecules, at least about 100,000 nucleic acidmolecules, at least about 500,000 nucleic acids, at least about1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acidmolecules, at least about 10,000,000 nucleic acid molecules, at leastabout 50,000,000 nucleic acid molecules, at least about 100,000,000nucleic acid molecules, at least about 250,000,000 nucleic acidmolecules and in some cases at least about 1 billion nucleic acidmolecules, or more. Nucleic acid molecules of a given bead can includeidentical (or common) barcode sequences, different barcode sequences, ora combination of both. Nucleic acid molecules of a given bead caninclude multiple sets of nucleic acid molecules. Nucleic acid moleculesof a given set can include identical barcode sequences. The identicalbarcode sequences can be different from barcode sequences of nucleicacid molecules of another set.

Moreover, when the population of beads is partitioned, the resultingpopulation of partitions can also include a diverse barcode library thatincludes at least about 1,000 different barcode sequences, at leastabout 5,000 different barcode sequences, at least about 10,000 differentbarcode sequences, at least at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences. Additionally, each partition of the population caninclude at least about 1,000 nucleic acid molecules, at least about5,000 nucleic acid molecules, at least about 10,000 nucleic acidmolecules, at least about 50,000 nucleic acid molecules, at least about100,000 nucleic acid molecules, at least about 500,000 nucleic acids, atleast about 1,000,000 nucleic acid molecules, at least about 5,000,000nucleic acid molecules, at least about 10,000,000 nucleic acidmolecules, at least about 50,000,000 nucleic acid molecules, at leastabout 100,000,000 nucleic acid molecules, at least about 250,000,000nucleic acid molecules and in some cases at least about 1 billionnucleic acid molecules.

In some cases, it may be desirable to incorporate multiple differentbarcodes within a given partition, either attached to a single ormultiple beads within the partition. For example, in some cases, amixed, but known set of barcode sequences may provide greater assuranceof identification in the subsequent processing, e.g., by providing astronger address or attribution of the barcodes to a given partition, asa duplicate or independent confirmation of the output from a givenpartition.

The nucleic acid molecules (e.g., oligonucleotides) are releasable fromthe beads upon the application of a particular stimulus to the beads. Insome cases, the stimulus may be a photo-stimulus, e.g., through cleavageof a photo-labile linkage that releases the nucleic acid molecules. Inother cases, a thermal stimulus may be used, where elevation of thetemperature of the beads environment will result in cleavage of alinkage or other release of the nucleic acid molecules form the beads.In still other cases, a chemical stimulus can be used that cleaves alinkage of the nucleic acid molecules to the beads, or otherwise resultsin release of the nucleic acid molecules from the beads. In one case,such compositions include the polyacrylamide matrices described abovefor encapsulation of analyte carriers, and may be degraded for releaseof the attached nucleic acid molecules through exposure to a reducingagent, such as DTT.

In some aspects, provided are systems and methods for controlledpartitioning. Droplet size may be controlled by adjusting certaingeometric features in channel architecture (e.g., microfluidics channelarchitecture). For example, an expansion angle, width, and/or length ofa channel may be adjusted to control droplet size.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets. A channelstructure 400 can include a channel segment 402 communicating at achannel junction 406 (or intersection) with a reservoir 404. Thereservoir 404 can be a chamber. Any reference to “reservoir,” as usedherein, can also refer to a “chamber.” In operation, an aqueous fluid408 that includes suspended beads 412 may be transported along thechannel segment 402 into the junction 406 to meet a second fluid 410that is immiscible with the aqueous fluid 408 in the reservoir 404 tocreate droplets 416, 418 of the aqueous fluid 408 flowing into thereservoir 404. At the junction 406 where the aqueous fluid 408 and thesecond fluid 410 meet, droplets can form based on factors such as thehydrodynamic forces at the junction 406, flow rates of the two fluids408, 410, fluid properties, and certain geometric parameters (e.g., w,h₀, α, etc.) of the channel structure 400. A plurality of droplets canbe collected in the reservoir 404 by continuously injecting the aqueousfluid 408 from the channel segment 402 through the junction 406.

A discrete droplet generated may include a bead (e.g., as in occupieddroplets 416). Alternatively, a discrete droplet generated may includemore than one bead. Alternatively, a discrete droplet generated may notinclude any beads (e.g., as in unoccupied droplet 418). In someinstances, a discrete droplet generated may contain one or more analytecarriers, as described elsewhere herein. In some instances, a discretedroplet generated may comprise one or more reagents, as describedelsewhere herein.

In some instances, the aqueous fluid 408 can have a substantiallyuniform concentration or frequency of beads 412. The beads 412 can beintroduced into the channel segment 402 from a separate channel (notshown in FIG. 4). The frequency of beads 412 in the channel segment 402may be controlled by controlling the frequency in which the beads 412are introduced into the channel segment 402 and/or the relative flowrates of the fluids in the channel segment 402 and the separate channel.In some instances, the beads can be introduced into the channel segment402 from a plurality of different channels, and the frequency controlledaccordingly.

In some instances, the aqueous fluid 408 in the channel segment 402 cancomprise biological particles (or analyte carriers, e.g., described withreference to FIGS. 1 and 2). In some instances, the aqueous fluid 408can have a substantially uniform concentration or frequency ofbiological particles. As with the beads, the biological particles can beintroduced into the channel segment 402 from a separate channel. Thefrequency or concentration of the biological particles in the aqueousfluid 408 in the channel segment 402 may be controlled by controllingthe frequency in which the biological particles are introduced into thechannel segment 402 and/or the relative flow rates of the fluids in thechannel segment 402 and the separate channel. In some instances, thebiological particles can be introduced into the channel segment 402 froma plurality of different channels, and the frequency controlledaccordingly. In some instances, a first separate channel can introducebeads and a second separate channel can introduce biological particlesinto the channel segment 402. The first separate channel introducing thebeads may be upstream or downstream of the second separate channelintroducing the biological particles.

The second fluid 410 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resultingdroplets.

In some instances, the second fluid 410 may not be subjected to and/ordirected to any flow in or out of the reservoir 404. For example, thesecond fluid 410 may be substantially stationary in the reservoir 404.In some instances, the second fluid 410 may be subjected to flow withinthe reservoir 404, but not in or out of the reservoir 404, such as viaapplication of pressure to the reservoir 404 and/or as affected by theincoming flow of the aqueous fluid 408 at the junction 406.Alternatively, the second fluid 410 may be subjected and/or directed toflow in or out of the reservoir 404. For example, the reservoir 404 canbe a channel directing the second fluid 410 from upstream to downstream,transporting the generated droplets.

The channel structure 400 at or near the junction 406 may have certaingeometric features that at least partly determine the sizes of thedroplets formed by the channel structure 400. The channel segment 402can have a height, h₀ and width, w, at or near the junction 406. By wayof example, the channel segment 402 can comprise a rectangularcross-section that leads to a reservoir 404 having a wider cross-section(such as in width or diameter). Alternatively, the cross-section of thechannel segment 402 can be other shapes, such as a circular shape,trapezoidal shape, polygonal shape, or any other shapes. The top andbottom walls of the reservoir 404 at or near the junction 406 can beinclined at an expansion angle, α. The expansion angle, a, allows thetongue (portion of the aqueous fluid 408 leaving channel segment 402 atjunction 406 and entering the reservoir 404 before droplet formation) toincrease in depth and facilitate decrease in curvature of theintermediately formed droplet. Droplet size may decrease with increasingexpansion angle. The resulting droplet radius, R_(d), may be predictedby the following equation for the aforementioned geometric parameters ofh₀, w, and a:

$R_{d} \approx {0.44\left( {1 + {2.2\sqrt{\tan \; \alpha}\frac{w}{h_{0}}}} \right)\frac{h_{0}}{\sqrt{\tan \; \alpha}}}$

By way of example, for a channel structure with w=21 μm, h=21 μm, andα=3°, the predicted droplet size is 121 μm. In another example, for achannel structure with w=25 μm, h=25 μm, and α=5°, the predicted dropletsize is 123 μm. In another example, for a channel structure with w=28μm, h=28 μm, and α=7°, the predicted droplet size is 124 μm.

In some instances, the expansion angle, a, may be between a range offrom about 0.5° to about 4°, from about 0.1° to about 10°, or from about00 to about 90°. For example, the expansion angle can be at least about0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°,4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°,55°, 60°, 65°, 70°, 75°, 80°, 85°, or higher. In some instances, theexpansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°,82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°,20°, 15°, 10°, 90°, 8°, 7°, 6°, 5°, 4°, 30°, 2°, 1°, 0.1°, 0.01°, orless. In some instances, the width, w, can be between a range of fromabout 100 micrometers (μm) to about 500 μm. In some instances, thewidth, w, can be between a range of from about 10 μm to about 200 μm.Alternatively, the width can be less than about 10 μm. Alternatively,the width can be greater than about 500 μm. In some instances, the flowrate of the aqueous fluid 408 entering the junction 406 can be betweenabout 0.04 microliters (μL)/minute (min) and about 40 μL/min. In someinstances, the flow rate of the aqueous fluid 408 entering the junction406 can be between about 0.01 microliters (μL)/minute (min) and about100 μL/min. Alternatively, the flow rate of the aqueous fluid 408entering the junction 406 can be less than about 0.01 μL/min.Alternatively, the flow rate of the aqueous fluid 408 entering thejunction 406 can be greater than about 40 μL/min, such as 45 L/min, 50μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75 μL/min, 80μL/min, 85 μL/min, 90 μL/min, 95 L/min, 100 μL/min, 110 L/min, 120μL/min, 130 μL/min, 140 μL/min, 150 μL/min, or greater. At lower flowrates, such as flow rates of about less than or equal to 10microliters/minute, the droplet radius may not be dependent on the flowrate of the aqueous fluid 408 entering the junction 406.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

The throughput of droplet generation can be increased by increasing thepoints of generation, such as increasing the number of junctions (e.g.,junction 406) between aqueous fluid 408 channel segments (e.g., channelsegment 402) and the reservoir 404. Alternatively or in addition, thethroughput of droplet generation can be increased by increasing the flowrate of the aqueous fluid 408 in the channel segment 402.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 500 can comprise a plurality of channel segments 502 and areservoir 504. Each of the plurality of channel segments 502 may be influid communication with the reservoir 504. The channel structure 500can comprise a plurality of channel junctions 506 between the pluralityof channel segments 502 and the reservoir 504. Each channel junction canbe a point of droplet generation. The channel segment 402 from thechannel structure 400 in FIG. 4 and any description to the componentsthereof may correspond to a given channel segment of the plurality ofchannel segments 502 in channel structure 500 and any description to thecorresponding components thereof. The reservoir 404 from the channelstructure 400 and any description to the components thereof maycorrespond to the reservoir 504 from the channel structure 500 and anydescription to the corresponding components thereof.

Each channel segment of the plurality of channel segments 502 maycomprise an aqueous fluid 508 that includes suspended beads 512. Thereservoir 504 may comprise a second fluid 510 that is immiscible withthe aqueous fluid 508. In some instances, the second fluid 510 may notbe subjected to and/or directed to any flow in or out of the reservoir504. For example, the second fluid 510 may be substantially stationaryin the reservoir 504. In some instances, the second fluid 510 may besubjected to flow within the reservoir 504, but not in or out of thereservoir 504, such as via application of pressure to the reservoir 504and/or as affected by the incoming flow of the aqueous fluid 508 at thejunctions. Alternatively, the second fluid 510 may be subjected and/ordirected to flow in or out of the reservoir 504. For example, thereservoir 504 can be a channel directing the second fluid 510 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 508 that includes suspended beads 512may be transported along the plurality of channel segments 502 into theplurality of junctions 506 to meet the second fluid 510 in the reservoir504 to create droplets 516, 518. A droplet may form from each channelsegment at each corresponding junction with the reservoir 504. At thejunction where the aqueous fluid 508 and the second fluid 510 meet,droplets can form based on factors such as the hydrodynamic forces atthe junction, flow rates of the two fluids 508, 510, fluid properties,and certain geometric parameters (e.g., w, h₀, α, etc.) of the channelstructure 500, as described elsewhere herein. A plurality of dropletscan be collected in the reservoir 504 by continuously injecting theaqueous fluid 508 from the plurality of channel segments 502 through theplurality of junctions 506. Throughput may significantly increase withthe parallel channel configuration of channel structure 500. Forexample, a channel structure having five inlet channel segmentscomprising the aqueous fluid 508 may generate droplets five times asfrequently than a channel structure having one inlet channel segment,provided that the fluid flow rate in the channel segments aresubstantially the same. The fluid flow rate in the different inletchannel segments may or may not be substantially the same. A channelstructure may have as many parallel channel segments as is practical andallowed for the size of the reservoir. For example, the channelstructure may have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 500, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 1500, 5000 or more parallel or substantiallyparallel channel segments.

The geometric parameters, w, h₀, and α, may or may not be uniform foreach of the channel segments in the plurality of channel segments 502.For example, each channel segment may have the same or different widthsat or near its respective channel junction with the reservoir 504. Forexample, each channel segment may have the same or different height ator near its respective channel junction with the reservoir 504. Inanother example, the reservoir 504 may have the same or differentexpansion angle at the different channel junctions with the plurality ofchannel segments 502. When the geometric parameters are uniform,beneficially, droplet size may also be controlled to be uniform evenwith the increased throughput. In some instances, when it is desirableto have a different distribution of droplet sizes, the geometricparameters for the plurality of channel segments 502 may be variedaccordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 600 can comprise a plurality of channel segments 602 arrangedgenerally circularly around the perimeter of a reservoir 604. Each ofthe plurality of channel segments 602 may be in fluid communication withthe reservoir 604. The channel structure 600 can comprise a plurality ofchannel junctions 606 between the plurality of channel segments 602 andthe reservoir 604. Each channel junction can be a point of dropletgeneration. The channel segment 402 from the channel structure 400 inFIG. 4 and any description to the components thereof may correspond to agiven channel segment of the plurality of channel segments 602 inchannel structure 600 and any description to the correspondingcomponents thereof. The reservoir 404 from the channel structure 400 andany description to the components thereof may correspond to thereservoir 604 from the channel structure 600 and any description to thecorresponding components thereof.

Each channel segment of the plurality of channel segments 602 maycomprise an aqueous fluid 608 that includes suspended beads 612. Thereservoir 604 may comprise a second fluid 610 that is immiscible withthe aqueous fluid 608. In some instances, the second fluid 610 may notbe subjected to and/or directed to any flow in or out of the reservoir604. For example, the second fluid 610 may be substantially stationaryin the reservoir 604. In some instances, the second fluid 610 may besubjected to flow within the reservoir 604, but not in or out of thereservoir 604, such as via application of pressure to the reservoir 604and/or as affected by the incoming flow of the aqueous fluid 608 at thejunctions. Alternatively, the second fluid 610 may be subjected and/ordirected to flow in or out of the reservoir 604. For example, thereservoir 604 can be a channel directing the second fluid 610 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 608 that includes suspended beads 612may be transported along the plurality of channel segments 602 into theplurality of junctions 606 to meet the second fluid 610 in the reservoir604 to create a plurality of droplets 616. A droplet may form from eachchannel segment at each corresponding junction with the reservoir 604.At the junction where the aqueous fluid 608 and the second fluid 610meet, droplets can form based on factors such as the hydrodynamic forcesat the junction, flow rates of the two fluids 608, 610, fluidproperties, and certain geometric parameters (e.g., widths and heightsof the channel segments 602, expansion angle of the reservoir 604, etc.)of the channel structure 600, as described elsewhere herein. A pluralityof droplets can be collected in the reservoir 604 by continuouslyinjecting the aqueous fluid 608 from the plurality of channel segments602 through the plurality of junctions 606. Throughput may significantlyincrease with the substantially parallel channel configuration of thechannel structure 600. A channel structure may have as manysubstantially parallel channel segments as is practical and allowed forby the size of the reservoir. For example, the channel structure mayhave at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1000, 1500, 5000 or more parallel or substantially parallel channelsegments. The plurality of channel segments may be substantially evenlyspaced apart, for example, around an edge or perimeter of the reservoir.Alternatively, the spacing of the plurality of channel segments may beuneven.

The reservoir 604 may have an expansion angle, a (not shown in FIG. 6)at or near each channel junction. Each channel segment of the pluralityof channel segments 602 may have a width, w, and a height, h₀, at ornear the channel junction. The geometric parameters, w, h₀, and α, mayor may not be uniform for each of the channel segments in the pluralityof channel segments 602. For example, each channel segment may have thesame or different widths at or near its respective channel junction withthe reservoir 604. For example, each channel segment may have the sameor different height at or near its respective channel junction with thereservoir 604.

The reservoir 604 may have the same or different expansion angle at thedifferent channel junctions with the plurality of channel segments 602.For example, a circular reservoir (as shown in FIG. 6) may have aconical, dome-like, or hemispherical ceiling (e.g., top wall) to providethe same or substantially same expansion angle for each channel segments602 at or near the plurality of channel junctions 606. When thegeometric parameters are uniform, beneficially, resulting droplet sizemay be controlled to be uniform even with the increased throughput. Insome instances, when it is desirable to have a different distribution ofdroplet sizes, the geometric parameters for the plurality of channelsegments 602 may be varied accordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size. The beads and/or analytecarrier injected into the droplets may or may not have uniform size.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.A channel structure 700 can include a channel segment 702 communicatingat a channel junction 706 (or intersection) with a reservoir 704. Insome instances, the channel structure 700 and one or more of itscomponents can correspond to the channel structure 100 and one or moreof its components. FIG. 7B shows a perspective view of the channelstructure 700 of FIG. 7A.

An aqueous fluid 712 comprising a plurality of particles 716 may betransported along the channel segment 702 into the junction 706 to meeta second fluid 714 (e.g., oil, etc.) that is immiscible with the aqueousfluid 712 in the reservoir 704 to create droplets 720 of the aqueousfluid 712 flowing into the reservoir 704. At the junction 706 where theaqueous fluid 712 and the second fluid 714 meet, droplets can form basedon factors such as the hydrodynamic forces at the junction 706, relativeflow rates of the two fluids 712, 714, fluid properties, and certaingeometric parameters (e.g., Δh, etc.) of the channel structure 700. Aplurality of droplets can be collected in the reservoir 704 bycontinuously injecting the aqueous fluid 712 from the channel segment702 at the junction 706.

A discrete droplet generated may comprise one or more particles of theplurality of particles 716. As described elsewhere herein, a particlemay be any particle, such as a bead, cell bead, gel bead, analytecarrier, macromolecular constituents of analyte carrier, or otherparticles. Alternatively, a discrete droplet generated may not includeany particles.

In some instances, the aqueous fluid 712 can have a substantiallyuniform concentration or frequency of particles 716. As describedelsewhere herein (e.g., with reference to FIG. 4), the particles 716(e.g., beads) can be introduced into the channel segment 702 from aseparate channel (not shown in FIG. 7). The frequency of particles 716in the channel segment 702 may be controlled by controlling thefrequency in which the particles 716 are introduced into the channelsegment 702 and/or the relative flow rates of the fluids in the channelsegment 702 and the separate channel. In some instances, the particles716 can be introduced into the channel segment 702 from a plurality ofdifferent channels, and the frequency controlled accordingly. In someinstances, different particles may be introduced via separate channels.For example, a first separate channel can introduce beads and a secondseparate channel can introduce biological particles into the channelsegment 702. The first separate channel introducing the beads may beupstream or downstream of the second separate channel introducing thebiological particles.

In some instances, the second fluid 714 may not be subjected to and/ordirected to any flow in or out of the reservoir 704. For example, thesecond fluid 714 may be substantially stationary in the reservoir 704.In some instances, the second fluid 714 may be subjected to flow withinthe reservoir 704, but not in or out of the reservoir 704, such as viaapplication of pressure to the reservoir 704 and/or as affected by theincoming flow of the aqueous fluid 712 at the junction 706.Alternatively, the second fluid 714 may be subjected and/or directed toflow in or out of the reservoir 704. For example, the reservoir 704 canbe a channel directing the second fluid 714 from upstream to downstream,transporting the generated droplets.

The channel structure 700 at or near the junction 706 may have certaingeometric features that at least partly determine the sizes and/orshapes of the droplets formed by the channel structure 700. The channelsegment 702 can have a first cross-section height, h₁, and the reservoir704 can have a second cross-section height, h₂. The first cross-sectionheight, h₁, and the second cross-section height, h₂, may be different,such that at the junction 706, there is a height difference of Δh. Thesecond cross-section height, h₂, may be greater than the firstcross-section height, h₁. In some instances, the reservoir maythereafter gradually increase in cross-section height, for example, themore distant it is from the junction 706. In some instances, thecross-section height of the reservoir may increase in accordance withexpansion angle, β, at or near the junction 706. The height difference,Δh, and/or expansion angle, β, can allow the tongue (portion of theaqueous fluid 712 leaving channel segment 702 at junction 706 andentering the reservoir 704 before droplet formation) to increase indepth and facilitate decrease in curvature of the intermediately formeddroplet. For example, droplet size may decrease with increasing heightdifference and/or increasing expansion angle.

The height difference, Δh, can be at least about 1 μm. Alternatively,the height difference can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 200, 300, 400, 500 μm or more. Alternatively, theheight difference can be at most about 500, 400, 300, 200, 100, 90, 80,70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, 1 μm or less. In some instances, theexpansion angle, β, may be between a range of from about 0.5° to about4°, from about 0.1° to about 10°, or from about 0° to about 90°. Forexample, the expansion angle can be at least about 0.01°, 0.1°, 0.2°,0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°,8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°,75°, 80°, 85°, or higher. In some instances, the expansion angle can beat most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°,70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°,7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.

In some instances, the flow rate of the aqueous fluid 712 entering thejunction 706 can be between about 0.04 microliters (μL)/minute (min) andabout 40 μL/min. In some instances, the flow rate of the aqueous fluid712 entering the junction 706 can be between about 0.01 microliters(μL)/minute (min) and about 100 μL/min. Alternatively, the flow rate ofthe aqueous fluid 712 entering the junction 706 can be less than about0.01 μL/min. Alternatively, the flow rate of the aqueous fluid 712entering the junction 706 can be greater than about 40 μL/min, such as45 μL/min, 50 μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75μL/min, 80 μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110μL/min, 120 μL/min, 130 μL/min, 140 μL/min, 150 μL/min, or greater. Atlower flow rates, such as flow rates of about less than or equal to 10microliters/minute, the droplet radius may not be dependent on the flowrate of the aqueous fluid 712 entering the junction 706. The secondfluid 714 may be stationary, or substantially stationary, in thereservoir 704. Alternatively, the second fluid 714 may be flowing, suchas at the above flow rates described for the aqueous fluid 712.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

While FIGS. 7A and 7B illustrate the height difference, Δh, being abruptat the junction 706 (e.g., a step increase), the height difference mayincrease gradually (e.g., from about 0 μm to a maximum heightdifference). Alternatively, the height difference may decrease gradually(e.g., taper) from a maximum height difference. A gradual increase ordecrease in height difference, as used herein, may refer to a continuousincremental increase or decrease in height difference, wherein an anglebetween any one differential segment of a height profile and animmediately adjacent differential segment of the height profile isgreater than 90°. For example, at the junction 706, a bottom wall of thechannel and a bottom wall of the reservoir can meet at an angle greaterthan 90°. Alternatively or in addition, a top wall (e.g., ceiling) ofthe channel and a top wall (e.g., ceiling) of the reservoir can meet anangle greater than 90°. A gradual increase or decrease may be linear ornon-linear (e.g., exponential, sinusoidal, etc.). Alternatively or inaddition, the height difference may variably increase and/or decreaselinearly or non-linearly. While FIGS. 7A and 7B illustrate the expandingreservoir cross-section height as linear (e.g., constant expansionangle, β), the cross-section height may expand non-linearly. Forexample, the reservoir may be defined at least partially by a dome-like(e.g., hemispherical) shape having variable expansion angles. Thecross-section height may expand in any shape.

The channel networks, e.g., as described above or elsewhere herein, canbe fluidly coupled to appropriate fluidic components. For example, theinlet channel segments are fluidly coupled to appropriate sources of thematerials they are to deliver to a channel junction. These sources mayinclude any of a variety of different fluidic components, from simplereservoirs defined in or connected to a body structure of a microfluidicdevice, to fluid conduits that deliver fluids from off-device sources,manifolds, fluid flow units (e.g., actuators, pumps, compressors) or thelike. Likewise, the outlet channel segment (e.g., channel segment 208,reservoir 604, etc.) may be fluidly coupled to a receiving vessel orconduit for the partitioned cells for subsequent processing. Again, thismay be a reservoir defined in the body of a microfluidic device, or itmay be a fluidic conduit for delivering the partitioned cells to asubsequent process operation, instrument or component.

The methods and systems described herein may be used to greatly increasethe efficiency of single cell applications and/or other applicationsreceiving droplet-based input. For example, following the sorting ofoccupied cells and/or appropriately-sized cells, subsequent operationsthat can be performed can include generation of amplification products,purification (e.g., via solid phase reversible immobilization (SPRI)),further processing (e.g., shearing, ligation of functional sequences,and subsequent amplification (e.g., via PCR)). These operations mayoccur in bulk (e.g., outside the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken andthe contents of the droplet pooled for additional operations. Additionalreagents that may be co-partitioned along with the barcode bearing beadmay include oligonucleotides to block ribosomal RNA (rRNA) and nucleasesto digest genomic DNA from cells. Alternatively, rRNA removal agents maybe applied during additional processing operations. The configuration ofthe constructs generated by such a method can help minimize (or avoid)sequencing of the poly-T sequence during sequencing and/or sequence the5′ end of a polynucleotide sequence. The amplification products, forexample, first amplification products and/or second amplificationproducts, may be subject to sequencing for sequence analysis. In somecases, amplification may be performed using the Partial HairpinAmplification for Sequencing (PHASE) method.

A variety of applications require the evaluation of the presence andquantification of different analyte carrier or organism types within apopulation of analyte carriers, including, for example, microbiomeanalysis and characterization, environmental testing, food safetytesting, epidemiological analysis, e.g., in tracing contamination or thelike.

Computer Systems

The present disclosure provides computer systems that are programmed toimplement methods of the disclosure. FIG. 21 shows a computer system2101 that is programmed or otherwise configured to (i) control amicrofluidics system (e.g., fluid flow), (ii) sort occupied dropletsfrom unoccupied droplets, (iii) polymerize droplets, (iv) performsequencing applications, (v) generate and maintain a library of barcodebeads, and (vi) analyze nucleic acid sequences. The computer system 2101can regulate various aspects of the present disclosure, such as, forexample, regulating fluid flow rate in one or more channels in amicrofluidic structure and regulating polymerization application units.The computer system 2101 can be an electronic device of a user or acomputer system that is remotely located with respect to the electronicdevice. The electronic device can be a mobile electronic device.

The computer system 2101 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 2105, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 2101 also includes memory or memorylocation 2110 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 2115 (e.g., hard disk), communicationinterface 2120 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 2125, such as cache, othermemory, data storage and/or electronic display adapters. The memory2110, storage unit 2115, interface 2120 and peripheral devices 2125 arein communication with the CPU 2105 through a communication bus (solidlines), such as a motherboard. The storage unit 2115 can be a datastorage unit (or data repository) for storing data. The computer system2101 can be operatively coupled to a computer network (“network”) 2130with the aid of the communication interface 2120. The network 2130 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 2130 insome cases is a telecommunication and/or data network. The network 2130can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 2130, in some cases withthe aid of the computer system 2101, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 2101 tobehave as a client or a server.

The CPU 2105 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 2110. The instructionscan be directed to the CPU 2105, which can subsequently program orotherwise configure the CPU 2105 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 2105 can includefetch, decode, execute, and writeback.

The CPU 2105 can be part of a circuit, such as an integrated circuit.One or more other components of the system 2101 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 2115 can store files, such as drivers, libraries andsaved programs. The storage unit 2115 can store user data, e.g., userpreferences and user programs. The computer system 2101 in some casescan include one or more additional data storage units that are externalto the computer system 2101, such as located on a remote server that isin communication with the computer system 2101 through an intranet orthe Internet.

The computer system 2101 can communicate with one or more remotecomputer systems through the network 2130. For instance, the computersystem 2101 can communicate with a remote computer system of a user(e.g., operator). Examples of remote computer systems include personalcomputers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad,Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone,Android-enabled device, Blackberry®), or personal digital assistants.The user can access the computer system 2101 via the network 2130.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 2101, such as, for example, on thememory 2110 or electronic storage unit 2115. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 2105. In some cases, thecode can be retrieved from the storage unit 2115 and stored on thememory 2110 for ready access by the processor 2105. In some situations,the electronic storage unit 2115 can be precluded, andmachine-executable instructions are stored on memory 2110.

The code can be pre-compiled and configured for use with a machinehaving a processor adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 2101, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 2101 can include or be in communication with anelectronic display 2135 that comprises a user interface (UI) 2140 forproviding, for example, results of sequencing analysis. Examples of UIsinclude, without limitation, a graphical user interface (GUI) andweb-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 2105. Thealgorithm can, for example, perform sequencing and analyze sequencingresults.

EXAMPLES Example 1: Single Cell Epigenetic Profiling

Cells are isolated from a subject. The nuclei from the cells areisolated, permeabilized, and partitioned into droplets comprisingpolymeric precursors, such that each droplet comprises at most onenucleus. The droplets also comprise magnetic particles. The polymericprecursors are subjected to conditions sufficient to polymerize theprecursors, such that cell beads are generated each comprising a singlepermeabilized nucleus and at least one magnetic particle. Cell beads areplaced in an aqueous solution comprising an antibody and an engineeredMNase covalently linked to Protein A. The engineered MNase comprises abiotin molecule on its surface near the active site of the enzyme. Theantibody is capable of binding Histone H2A protein. The antibodymolecules are allowed to bind to the Histone H2A proteins in the nucleiof the cell beads, and the engineered MNase attached to Protein A isallowed to bind to the antibody molecules. Unbound antibody andengineered MNase is removed by washing.

The cell beads comprising the antibody and engineered MNase bound to theHistone H2A proteins are partitioned into a droplet together with abarcode bead in an aqueous solution comprising Mg²⁺, Ca²⁺, a ligaseenzyme, and a functionalized dextran polymer. The barcode bead comprisesY-adapter nucleic acid barcode molecules attached thereto. Thefunctionalized dextran polymer is attached to streptavidin molecules.The Mg²⁺ and Ca²⁺ diffuse into the cell bead, thereby activating theengineered MNase. The engineered MNase cleaves the genomic DNA in thechromatin of the nucleus near Histone H2A proteins, thereby generatingDNA fragments. The DNA fragments and the engineered MNase diffuse out ofthe cell bead into the droplet. The biotin molecule on the engineeredMNase binds to the streptavidin molecule on the functionalized dextranpolymer, thereby inactivating the engineered MNase.

The Y-adapter nucleic acid barcode molecules are released from thebarcode bead into the droplet. The ligase enzyme ligates the Y-adapternucleic acid barcode molecules onto the DNA fragments, generatingbarcoded DNA fragments. The barcoded DNA fragments and cell bead arereleased from the droplet into a solution, and the cell bead is removedfrom the solution by magnetic purification. The barcoded DNA fragmentsare purified and subjected to nucleic acid sequencing to generatenucleic acid sequences from a single cell. Analysis of these nucleicacid sequences is performed to generate an epigenetic profile of thesingle cell.

Devices, systems, compositions and methods of the present disclosure maybe used for various applications, such as, for example, processing asingle analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g.,DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein)from a single cell. For example, a analyte carrier (e.g., a cell or cellbead) is partitioned in a partition (e.g., droplet), and multipleanalytes from the analyte carrier are processed for subsequentprocessing. The multiple analytes may be from the single cell. This mayenable, for example, simultaneous proteomic, transcriptomic and genomicanalysis of the cell.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method for nucleic acid analysis, comprising:(a) generating a partition comprising (i) a cell bead comprising anucleic acid molecule, (ii) a nucleic acid barcode molecule, and (iii) apolymer; and (b) using said nucleic acid molecule or a derivativethereof and said nucleic acid barcode molecule to perform one or morereactions in said partition to generate a barcoded nucleic acidmolecule.
 2. The method of claim 1, wherein said partition is a dropletor well.
 3. (canceled)
 4. The method of claim 1, wherein said one ormore reactions are performed outside said cell bead.
 5. The method ofclaim 1, wherein said one or more reactions comprise nucleic acidextension, nucleic acid amplification, or nucleic acid ligation. 6.(canceled)
 7. (canceled)
 8. The method of claim 1, wherein said cellbead or said polymer is functionalized.
 9. The method of claim 1,wherein said polymer is a positively charged or a negatively chargedpolymer.
 10. (canceled)
 11. (canceled)
 12. The method of claim 1,wherein said cell bead or said polymer is attached to a reagent.
 13. Themethod of claim 12, wherein said reagent is an enzyme, a nucleic acidmolecule comprising a capture sequence, an aptamer, a chemical compound,or a polypeptide.
 14. The method of claim 12, wherein said reagentcomprises biotin, a small molecule inhibitor, or a chelating agent.15.-17. (canceled)
 18. The method of claim 14, wherein said chelatingagent is ethylenediaminetetraacetic acid (EDTA) or ethyleneglycol-bis(P-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). 19.The method of claim 13, wherein said capture sequence is a poly-thymine(poly-T) sequence, a random N-mer sequence, or a targeted capturesequence.
 20. The method of claim 13, wherein said enzyme is a nuclease,transposase, polymerase, helicase, reverse transcriptase, ligase, orphosphatase.
 21. The method of claim 13, wherein said nucleic acidmolecule comprising said capture sequence further comprises one or morefunctional sequences selected from the group consisting of a barcodesequence, a unique molecular index (UMI) sequence, a sequencing primersequence, a partial sequencing primer sequence, and a sequenceconfigured to attach to the flow cell of a sequencer.
 22. The method ofclaim 1, wherein said partition further comprises an enzyme configuredto perform said one or more reactions on said nucleic acid molecule or aderivative thereof.
 23. The method of claim 22, wherein said partitioncomprises a cation for activating said enzyme.
 24. The method of claim23, wherein said cation is magnesium or calcium.
 25. (canceled)
 26. Themethod of claim 22, wherein said enzyme is a nuclease, transposase,polymerase, helicase, reverse transcriptase, ligase, or phosphatase. 27.The method of claim 26, wherein said nuclease is micrococcal nuclease(MNase) or a deoxyribonuclease (DNase).
 28. (canceled)
 29. The method ofclaim 26, further comprising, prior to (b), using said nuclease tocleave said nucleic acid molecule to generate a nucleic acid fragment.30. The method of 22, wherein said enzyme is an engineered enzyme. 31.(canceled)
 32. The method of claim 30, wherein said engineered enzyme isconfigured to bind to a binding partner wherein said binding partnerinhibits the activity of said engineered enzyme. 33.-38. (canceled) 39.The method of claim 1, wherein said nucleic acid barcode molecule isattached to a bead.
 40. (canceled)
 41. (canceled)
 42. The method ofclaim 39, wherein said bead is a gel bead. 43.-45. (canceled)
 46. Themethod of claim 1, wherein said polymer is a linear or a branchedpolymer.
 47. (canceled)
 48. The method of claim 1, wherein said polymeris not cross-linked.
 49. The method of claim 1, wherein said polymer isdextran, polyethylene glycol (PEG), or polyacrylamide.
 50. The method ofclaim 1, wherein said partition further comprises a magnetic particle.51. (canceled)
 52. (canceled)
 53. The method of claim 50, wherein saidmagnetic particle comprises, attached thereto, a nucleic acid moleculecomprising a capture sequence.
 54. The method of claim 53, wherein saidcapture sequence is a poly-T sequence, a random N-mer sequence, or atargeted capture sequence.
 55. The method of claim 1, wherein saidpolymer is incapable of diffusing into said cell bead. 56.-143.(canceled)